Reduced byproduct high solids polyamine-epihalohydrin compositions

ABSTRACT

Processes for rendering a polyamine-epihalohydrin resin storage stable, including processes that prepare a storage stable resin and/or processes that treat resins. A composition containing a polyamine-epihalohydrin resin with a solids content of at least 15 wt % treated with at least one enzymatic agent under conditions to at least one of inhibit, reduce and remove the CPD-forming species to obtain a gelation storage stable reduced CPD-forming resin so that the composition containing the reduced CPD-forming polyamine-epihalohydrin resin when stored for 24 hours at 50° C., and a pH of about 1.0 releases less than about 250 ppm dry basis of CPD for wet strength polyamine-epihalohydrin resin and less than about 100 ppm dry basis of CPD for creping polyamine-epihalohydrin resin. A process for treating polyamine-epihalohydrin resin, to reduce the level of the nitrogen-free organohalogen compound by adding at least one microorganism, or at least one enzyme isolated from the at least one microorganism to an aqueous composition containing a solids content of at least 15 wt %, under conditions to dehalogenate the nitrogen-free organohalogen compound so as to reduce a level of the nitrogen-free organohalogen compound while leaving the polyamine-epihalohydrin resin substantially intact. A process for rendering a polyamine-epihalohydrin resin storage stable by treating the resin of less than 15 wt % with at least one enzymatic agent under conditions to at least one of inhibit, reduce and remove the CPD-forming species to obtain a gelation storage stable reduced CPD-forming resin that when stored for 24 hours at 50° C., and a pH of about 1.0 releases less than about 250 ppm dry basis while simultaneously treating the polyamine-epihalohydrin resin by contacting with at least one microorganism, or at least one enzyme isolated from the at least one microorganism, in an amount, and at a pH and temperature effective to dehalogenate residual quantities of organically bound halogen. A paper product containing the storage stable polyamine-epihalohydrin resin, when corrected for adding at about a 1 wt % addition level of the resin, contains less than about 250 ppb of CPD.

FIELD OF THE INVENTION

[0001] This invention relates to resins and aqueous compositionscontaining resins, and processes of forming resin compositions,especially for the paper industry, including strength agents, such aswet strength and dry strength agents, and creping agents. The presentinvention also relates to resins, as well as processes for theirproduction, wherein the resins, and compositions and products, such aspaper products, containing the resins have reduced residuals, such asepihalohydrins and epihalohydrin hydrolysis products. Still further, thepresent invention relates to resins, and compositions and products, suchas paper products, which maintain low levels of residuals, such asepihalohydrins and epihalohydrin hydrolysis products, when stored. Stillfurther, each aspect of the present invention relates to compositionshaving the resin at various solids contents, especially high solidscontents.

BACKGROUND OF THE INVENTION

[0002] Wet strength resins are often added to paper and paperboard atthe time of manufacture. In the absence of wet strength resins, papernormally retains only 3% to 5% of its strength after being wetted withwater. However, paper made with wet strength resin generally retains atleast 10%-50% of its strength when wet. Wet strength is useful in a widevariety of paper applications, some examples of which are toweling, milkand juice cartons, paper bags, and liner board for corrugatedcontainers.

[0003] Dry strength is also a critical paper property, particularly inlight of the recent trend for paper manufacturers to use high yield woodpulps in paper in order to achieve lower costs. These high yield woodpulps generally yield paper with significantly reduced strength whencompared to paper made from highly refined pulps.

[0004] Commercially available wet strength resins include Kymene®557H,Kymene® 557LX, Kymene® SLX, Kymene® Plus, Kymene® 450 and Kymene® 736wet strength resins, available from Hercules Incorporated, Wilmington,Del. Wet strength resins, such as those listed above, also provideincreased dry strength to paper.

[0005] Resins similar to those used for imparting strength to paper arealso often used as creping adhesives. In the manufacture of some paperproducts such as facial tissue, bathroom tissue, or paper towers, thepaper web is conventionally subjected to a creping process in order togive it desirable textural characteristics, such as softness and bulk.The creping process typically involves adhering the web, a cellulose webin the case of paper, to a rotating creping cylinder, such as theapparatus known as a Yankee dryer, and then dislodging the adhered webwith a doctor blade. The impact of the web against the doctor bladeruptures some of the fiber-to-fiber bonds within the web and causes theweb to wrinkle or pucker.

[0006] The severity of this creping action is dependent upon a number offactors, including the degree of adhesion between the web and thesurface of the creping cylinder. Greater adhesion causes increasesoftness, although generally with some loss of strength. In order toincrease adhesion, a creping adhesive may be used to enhance anynaturally occurring adhesion that the web may have due to its watercontent, which will vary widely depending on the extent to which the webhas been previously dried. Creping adhesives should also prevent wear ofthe dryer surface and provide lubrication between the doctor blade andthe dryer surface and reduce chemical corrosion, as well as controllingthe extent of creping. A creping adhesive coating that adheres the sheetjust tightly enough to the drum will give a good crepe, impartingabsorbance and softness with the least possible loss of paper strength.If adhesion to the dryer drum is too strong, the sheet may pick or even“plug”, i.e., underride the doctor blade, and wrap around the dryerdrum. If there is not enough adhesion, the sheet will lift off tooeasily and undergo too little creping.

[0007] The creping adhesive, usually as an aqueous solution ordispersion, is generally sprayed onto the surface of the crepingcylinder or drum, e.g., a Yankee dryer. This improves heat transfer,allowing more efficient drying of the sheet. If the pulp furnish stickstoo strongly to the creping cylinder, release agents can be sprayed onthe cylinder. The release agents are typically hydrocarbon oils. Theseagents aid in the uniform release of the tissue web at the crepingblade, and also lubricate and protect the blade from excessive wear.

[0008] Examples of creping adhesive compositions include those disclosedin U.S. Pat. No. 5,187,219 to Furman, which is incorporated by referenceherein in its entirety. The compositions comprise water-solubleglyoxylated acrylamide/diallyldimethyl-ammonium chloride polymer and awater-soluble polyol having a molecular weight below 3000 as aplasticizer for the polymer.

[0009] U.S. Pat. No. 5,246,544 to Hollenberg et al., which isincorporated by reference herein in its entirety, discloses a reversiblycrosslinked creping adhesive which contains a nonself-crosslinkablematerial that is a polymer or oligomer having functional groups that canbe crosslinked by ionic crosslinking and at least one metal, cationiccrosslinking agent having a valence of four or more. The adhesive canalso contain additives to modify the mechanical properties of thecrosslinked polymers, e.g., glycols, polyethylene glycols, and otherpolyols such as simple sugars and oligosaccharides.

[0010] Polyaminoamide/epichlorohydrin creping adhesives are disclosed inU.S. Pat. No. 5,338,807 to Espy et al., U.S. Pat. No. 5,994,449 toMaslanka, and Canadian Patent 979,579 Giles et al., which areincorporated by reference herein in their entireties.

[0011] U.S. Pat. No. 5,374,334 to Sommese et al., which is incorporatedby reference herein in its entirety, discloses a creping adhesive whichis a crosslinked vinyl amine/vinyl alcohol polymer containing from about1 to about 99% vinyl amine. Epichlorohydrin is disclosed as acrosslinking agent.

[0012] U.S. Pat. Nos. 4,684,439 and 4,788,243 to Soerens, which areincorporated by reference herein in their entireties, disclose crepingadhesives comprising mixtures of polyvinyl alcohol and water solublethermoplastic polyamide resin comprising the reaction product of apolyalkylenepolyamine, a saturated aliphatic dibasic carboxylic acid anda poly(oxyethylene) diamine.

[0013] In U.S. Pat. Nos. 4,501,640 and 4,528,316 to Soerens, which areincorporated by reference herein in their entireties, there is discloseda creping adhesive comprising a mixture of polyvinyl alcohol and a watersoluble, thermosetting cationic polyamide resin.

[0014] Commercially available creping adhesives include Crepetrol® 190,Crepetrol® 290, and Crepetrol® 80E cationic polymers, available fromHercules Incorporated, Wilmington, Del.

[0015] Moreover, polyamine-epihalohydrin resins, such aspolyaminopolyamide-epihalohydrin resins often contain large quantitiesof epihalohydrin hydrolysis products. For example, commercialpolyaminopolyamide-epichlorohydrin resins typically contain 1-10 wt %(dry basis) of the epichlorohydrin (epi) by-products,1,3-dichloropropanol (1,3-DCP), 2,3-dichloropropanol (2,3-DCP) and3-chloropropanediol (CPD). Epi by-products are also known as epiresiduals. Production of such resins with reduced levels of epiby-products has been the subject of much investigation. Environmentalpressures to produce resins with lower levels of adsorbable organichalogen (AOX) species have been increasing. “AOX” refers to theadsorbable organic halogen content of the resin, which can be determinedby means of adsorption onto carbon. AOX includes epichlorohydrin (epi)and epi by-products (1,3-dichloropropanol, 2,3-dichloropropanol and3-chloropropanediol) as well as organic halogen bound to the polymerbackbone.

[0016] Several ways of reducing the quantities of epihalohydrinhydrolysis products have been devised. Reduction in the quantity ofepihalohydrin used in the synthetic step is an alternative taught inU.S. Pat. No. 5,171,795. A longer reaction time results. Control overthe manufacturing process is taught in U.S. Pat. No. 5,017,642 to yieldcompositions of reduced concentration of hydrolysis products. Thesepatents are incorporated by reference herein in their entireties.

[0017] Post-synthesis treatments are also taught. U.S. Pat. No.5,256,727, which is incorporated by reference in its entirety, teachesthat reacting the epihalohydrin and its hydrolysis products with dibasicphosphate salts or alkanolamines in equimolar proportions converts thechlorinated organic compounds into non-chlorinated species. To do thisit is necessary to conduct a second reaction step for at least 3 hours,which adds significantly to costs and generates quantities of unwantedorganic or inorganic materials in the wet strength composition. Incompositions containing large amounts of epihalohydrin and epihalohydrinhydrolysis products (e.g., about 1-6% by weight of the composition), theamount of organic material formed is likewise present in undesirablylarge amounts.

[0018] U.S. Pat. No. 5,516,885 and WO 92/22601, which are incorporatedby reference in their entireties, disclose that halogenated by-productscan be removed from products containing high levels of halogenatedby-products as well as low levels of halogenated by-products by the useof ion exchange resins. However, it is clear from the data presentedthat there are significant yield losses in wet strength composition anda reduction in wet strength effectiveness.

[0019] It is known that nitrogen-free organohalogen-containing compoundscan be converted to a relatively harmless substance. For example,1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol (also known as3-chloropropanediol, 3-monochloropropanediol, monochloropropanediol,chloropropanediol, CPD, 3-CPD, MCPD and 3-MCPD) and epichlorohydrin havebeen treated with alkali to produce glycerol.

[0020] The conversion of nitrogen-free organohalogen compounds withmicroorganisms containing a dehalogenase is also known. For example, C.E. Castro, et al. (“Biological Cleavage of Carbon-Halogen BondsMetabolism of 3-Bromopropanol by Pseudomonas sp.”, Biochimica etBiophysica Acta, 100, 384-392, 1965), which is incorporated by referencein its entirety, describes the use of Pseudomonas sp. isolated from soilthat metabolizes 3-bromopropanol in sequence to 3-bromopropionic acid,3-hydroxypropionic acid and CO₂.

[0021] Various U.S. patents also describe the use of microorganisms fordehalogenating halohydrins, e.g., U.S. Pat. Nos. 4,452,894; 4,477,570;and 4,493,895. Each of these patents is hereby incorporated by referenceas though set forth in full herein.

[0022] U.S. Pat. Nos. 5,470,742, 5,843,763 and 5,871,616, which areincorporated by reference herein in their entireties, disclose the useof microorganisms or enzymes derived from microorganisms to removeepihalohydrin and epihalohydrin hydrolysis products from wet strengthcompositions without reduction in wet strength effectiveness.

[0023] U.S. application Ser. No. 09/629,629, filed Jul. 31, 2000, whichis incorporated by reference herein in its entirety, is directed to theuse of microorganisms or enzymes derived from microorganisms to removeepihalohydrin and epihalohydrin hydrolysis products from resincompositions, and discloses a preferred sequential method for growth ofthe microorganisms.

[0024] Still further, U.S. Pat. No. 5,972,691 and WO 96/40967, which areincorporated by reference in their entireties, disclose the treatment ofwet strength compositions with an inorganic base after the synthesisstep (i.e., after the polymerization reaction to form the resin) hasbeen completed and the resin has been stabilized at low pH, to reducethe organo halogen content of wet strength compositions (e.g.,chlorinated hydrolysis products) to moderate levels (e.g., about 0.5%based on the weight of the composition). The composition so formed canthen be treated with microorganisms or enzymes to economically producewet strength compositions with very low levels of epihalohydrins andepihalohydrin hydrolysis products.

[0025] It is also known that epihalohydrin and epihalohydrinhydrolyzates can be reacted with bases to form chloride ion andpolyhydric alcohols. U.S. Pat. No. 4,975,499 teaches the use of basesduring the synthetic step to reduce organo chlorine contents of wetstrength composition to moderate levels (e.g., to moderate levels offrom about 0.11 to about 0.16%) based on the weight of the composition.U.S. Pat. No. 5,019,606 teaches reacting wet strength compositions withan organic or inorganic base. These patents are incorporated byreference in their entireties.

[0026] Moreover, U.S. application Ser. Nos. 09/001,787, filed Dec. 31,1997, and 09/224,107, filed Dec. 22, 1998 to Riehle, and WO 99/33901,and which are incorporated by reference in their entireties, discloseamongst other features, a process for reducing the AOX content of astarting water-soluble wet-strength resin comprising azetidinium ionsand tertiary aminohalohydrin, which includes treating the resin inaqueous solution with base to form treated resin, wherein at least about20 mole % of the tertiary aminohalohydrin present in the starting resinis converted into epoxide and the level of azetidinium ion issubstantially unchanged, and the effectiveness of the treated resin inimparting wet strength is at least about as great as that of thestarting wet-strength resin.

[0027] Still further, U.S. Pat. application Ser. Nos. 09/592,681, filedJun. 12, 2000, 09/363,224, filed Jul. 30, 1999, 09/330,200, filed Jun.11, 1999, each of which is incorporated by reference in its entirety,are directed to polyamine-epihalohydrin resin products, particularlypolyamine-epihalohydrin resin products which can be stored with at leastreduced formation of halogen containing residuals, such as3-chloropropanediol (CPD). Moreover, these applications disclose the useof microorganisms or enzymes derived from microorganisms to removeepihalohydrin and epihalohydrin hydrolysis products from wet strengthcompositions without reduction in wet strength effectiveness.

[0028] WO 99/09252 describes thermosetting wet strength resins preparedfrom end-capped polyaminoamide polymers. The endcappers used aremonocarboxylic acids or monofunctional carboxylic esters, and are usedto control the molecular weight of the polyaminamide in order to obtainwet strength resins with a high solids content.

[0029] Each of the foregoing approaches has provided various results,and there has been a continuing need for improvement in the use ofpolyamine-epihalohydrin resin, especially at high solids content. Inparticular, there is still a need for resin compositions, such as wetstrength, dry strength and creping agent resins, that can be provided insolutions or dispersion of reasonable viscosity at relatively highpolymer solids concentrations. Thus, there is still a need for resinsthat can be prepared, stored, treated and transported as a dispersion orsolution containing high solids concentrations without productdeterioration from polymer crosslinking, such as gelation problems.

SUMMARY OF THE INVENTION

[0030] Enzyme treatment of tertiary amine-based resins can be carriedout at higher concentration than what was previously disclosed, when thecorrect balance of conditions of time, temperature pH and enzymeconcentration are utilized.

[0031] The present invention is directed to polyamine-epihalohydrinresin products, particularly polyamine-epihalohydrin resin productswhich can be stored with at least reduced formation of halogencontaining residuals, such as 3-chloropropanediol (CPD).

[0032] The present invention is also directed to various uses ofpolyamine-epihalohydrin resins having at least reduced formation ofhalogen containing residuals, such as strength agents, including wet anddry strength agents, and creping agents.

[0033] The present invention is also directed to polyamine-epihalohydrinresin products which have reduced levels of formation of CPD uponstorage, particularly paper products.

[0034] The present invention is also directed to various treatments ofpolyamine-epihalohydrin resins, including treatments to reduce theconcentration of halogen containing residuals associated with the resinsand/or compositions containing the resins.

[0035] The present invention is also directed to the preparation ofstorage stable polyamine-epihalohydrin resins and/or the treatment ofpolyamine-epihalohydrin resins to render such resins storage stable,especially at high solids concentrations.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Unless otherwise stated, all percentages, parts, ratios, etc.,are by weight.

[0037] Unless otherwise stated, a reference to a compound or componentincludes the compound or component by itself, as well as in combinationwith other compounds or components, such as mixtures of compounds.

[0038] Further, when an amount, concentration, or other value orparameter, is given as a list of upper preferable values and lowerpreferable values, this is to be understood as specifically disclosingall ranges formed from any pair of an upper preferred value and a lowerpreferred value, regardless whether ranges are separately disclosed.

[0039] U.S. Pat. application Ser. Nos. 09/592,681, filed Jun. 12, 2000,09/363,224, filed Jul. 30, 1999, and 09/330,200, filed Jun. 11, 1999,each of which is incorporated by reference in its entirety, are directedto the discovery that CPD that is formed in polyamine-epihalohydrinresins, after storage, is due to CPD-forming species that are associatedwith the oligomeric and/or polymeric component of the resin. It isdisclosed in these applications that polyamine-epihalohydrin resins canbe treated during and/or subsequent to production in such a manner so asto prevent the formation of, inhibit and/or remove elements associatedwith the polyamine-epihalohydrin resin which form CPD upon storage. Forexample, these applications disclose acid treatment, base treatment, lowacid endgroups in the prepolymer, and enzyme treatment to remove orreduce CPD-forming species.

[0040] Thus, in one aspect of the invention disclosed in U.S. Pat.application Ser. No. 09/592,681, polyamine-epihalohydrin resin productswhich have reduced levels of formation of CPD upon storage and minimizedlevels of CPD in paper products can be produced by treating the resinwith enzymatic agent. Thus, the CPD-forming species in the resin can bereduced and/or removed by treating the resin with an enzymatic agentthat is capable of releasing CPD-forming species from the resin. Theenzymatic agent can comprise one or more enzymes that are capable ofreleasing the CPD-forming species from the resin, such as at least oneof esterases, lipases and proteases. It is preferred that the enzymaticagent has esterase activity. One skilled in the art knows that theprotease class of enzymes can have esterase activity and that theesterase class of enzymes can have protease activity. A preferred classof proteases is the subtilisin group (E.C. 3.4.21.62. Homology modelingand protein engineering strategy of subtilases, the family ofsubtilisin-like serine proteinases, Siezen R J, de Vos W M, Leunissen JA, Dijkstra B W, Protein Eng. 1991, 4, 719-37), particularly the enzymesproduced from Bacillus licheniformis (Swiss-Prot Accession Number:P00780), Bacillus amyloliquifaciens (P00782), and Bacillus lentus(P29600). The enzyme can be in pure form, or the enzyme can beunpurified. Still further, mixtures of enzymes can be used, whichmixtures can include mixtures of pure enzymes, mixtures of unpurifiedenzymes, or mixtures of both. Particularly, preferred enzymatic agentsare ALCALASE and SAVINASE, which are obtainable from Novozymes NorthAmerica, Inc. Franklinton, N.C. (formerly known as Novo Nordisk Biochem,North America, Inc.).

[0041] Expanding upon the above, in previous work,polyamine-epichlorohydrin resins with about 12-13.5 wt % solids weretreated with ALCALASE 2.5 L type DX (Novozymes) to reduce or removeCPD-forming species. Under the treatment conditions, such as pH 8, 40°C., 6-8 hours and 0.25 g of ALCALASE for 30 g of resin, the resins had atendency to develop high viscosity and become unusable. It has beensurprisingly discovered in accordance with the present invention that bybalancing treatment conditions, including pH, temperature, concentrationof enzymatic agent, starting viscosity and solids concentration ofpolyamine-epihalohydrin resin containing compositions, such aspolyaminopolyamide-epichlorohydrin resin compositions, could be treatedwith enzymatic agent to reduce or remove CPD-forming species withdesired viscosity characteristics and excellent CPD release. These newlydiscovered conditions for enzymatic treatment allow the resin viscosityto be increased, decreased or maintained at the desired level, andpermit the enzymatic treatment at low solids contents, as well as highsolids concentrations of 15 wt % or greater.

[0042] Not wishing to be bound by theory, it is believed that as theactive solid content increases, the crosslinking rate increases andtherefore the viscosity increases. By judicious choice of reactionconditions, the rate of the crosslinking reaction that increasesviscosity can be balanced with the rate of the enzymatic hydrolysisreaction, which decreases viscosity, to predictably obtain desiredviscosity.

[0043] The present invention is useful because it enables higherthroughput production for the enzymatic treatment and because lowerlevels of the expensive enzyme can be used. This technology shouldtherefore enable (1) production of high solids, high effectivenessresins by allowing a longer time for azetidinium formation, and (2)production of lower AOX containing resins by increasing the conversionof tertiary aminochlorohydrin functionality to azetidiniumfunctionality.

[0044] Thus, according to the present invention, it has been discoveredthat enzyme treatment for reducing or removing CPD-forming species canbe performed at higher solids content of resin than would be expected.In this regard, the enzymatic treatment examples in the above-noted U.S.Pat. application Ser. No. 09/592,681 were performed at about 13-14 wt %solids. Thus, the enzyme treatment according to present invention caninclude solids contents as disclosed in the prior art, includingconcentrations as low as 4 wt % or lower. However, in contrast to theprior art, the solids content of the aqueous resin composition treatedwith enzymatic agent according to the present invention can be higherthan 15 wt %, more preferably higher than about 20 wt %, and can behigher than about 25 wt % especially with creping agents. Preferredsolids content ranges include from about 15 to 50 wt %, more preferablyabout 18 to 40 wt %. Preferably, for wet strength agents, the solidscontent is about 15 to 40 wt %, more preferably about 18 to 25 wt %,with one preferred solids value being about 21 wt %; and, for crepingagents, the solids content is about 20 to 40 wt %, more preferably about22 to 30 wt %, with one preferred solids value being about 26 wt %.

[0045] The terms creping aid, creping resin, creping agent and crepingadhesive are used interchangeably and all have the same meaningthroughout the specification.

[0046] The at least one enzymatic agent is added to the resin undersuitable conditions to achieve sufficient hydrolysis of CPD formingspecies in the high resin solids composition. Preferably, conditions oftime, temperature, pH, enzyme concentration, starting viscosity, andsolids content are balanced in order to enable the hydrolysis reactionwhile minimizing degradation of performance of the resin, such as wetstrength or creping effectiveness of the resin or preventing undesirablyhigh resin viscosity. Thus, unexpectedly the hydrolysis of CPD-formingspecies can be performed at high solids concentrations by balancing theconditions of time, temperature, pH, enzyme concentration, startingviscosity, and solids content. For example, as the solids concentrationincreases, the pH and/or temperature should usually be decreased.Moreover, as the solids concentration increases, the enzymeconcentration should usually be increased.

[0047] It is noted that the viscosity of the resin composition canincrease or decrease from a starting viscosity during enzymatictreatment, and it can remain the same or substantially the samedepending upon reaction conditions as noted above. For creping agents,it is usually preferred, but not limited to, that the viscosity at theend of the enzymatic treatment be the same or substantially the same asthe starting viscosity. For example, with wet strength agents, it isusually preferred, but not limited to, that the viscosity is maintainedor is decreased from the starting viscosity in the initial part of thetreatment time and then is maintained or increased to the desiredviscosity at the end of the treatment time. For example, with a resinhaving a starting Brookfield viscosity of about 100 to 300 cps and about20-22 wt % active solids, it is preferred that conditions are chosensuch that after treatment, the resin viscosity is maintained ordecreased with the active solids being about 19-22 wt %. Further forexample, with a resin having a starting Brookfield viscosity of about100 to 300 cps and about 20-22 wt % active solids, it is preferred ifthe Gardner Holdt viscosity at the beginning of the reaction is about Gto J, then it is desirable for the Gardner Holdt viscosity to decreaseduring the reaction to about F at the end of the reaction. Further forexample, with a resin having a starting Brookfield viscosity of about100 to 300 cps and about 20-22 wt % active solids, it is also preferredif the Gardner-Holt viscosity at the beginning of the reaction is aboutG to J, then it is desirable for the Gardner-Holt viscosity to decreaseduring the reaction to about A to E towards the end of the reaction, itis desirable to increase the treatment temperature until theGardner-Holt viscosity has increased to about F to I. Further forexample, with Kymene® E7219 (available from Hercules Incorporated,Wilmington, Del.) having a starting Brookfield viscosity about 200 to300 cps with about 20-22 wt % active solids if the Gardner-Holtviscosity at the beginning of the reaction is about I, then it isdesirable for the Gardner-Holt viscosity to decrease during the reactionto about F at the end of the reaction, resulting in a final resin(stabilized at about pH 3-3.5) with a Brookfield viscosity of about100-150 cps. For example, with Kymene® E7219 (available from HerculesIncorporated, Wilmington, Del.) having a starting Brookfield viscosityabout 200 to 300 cps with about 20-22 wt % active solids if theGardner-Holt viscosity at the beginning of the reaction is about I, thenit is desirable for the Gardner-Holt viscosity to decrease during thereaction to about C towards the end of the reaction, it is desirable toincrease the treatment temperature until the Gardner-Holt viscosity hasincreased to about F.

[0048] For example, with creping agents, it is usually preferred, butnot limited to, that the starting viscosity is below about 150 cps, morepreferably below about 100 cps, more preferably below about 80 cps andeven more preferably below about 40 cps. Preferably, the startingviscosity of the reaction mixture ranges from about 10 cps to 150 cpsmore preferably about 20 cps to 100 cps, and even more preferably about40 to 80 cps.

[0049] With respect to the above, it is preferred to minimize or atleast balance side reactions, such as polymeric breakdown or molecularweight increase in order that the viscosity of the reaction mixture isheld below a viscosity that would not enable the reaction to proceed.Preferably, viscosity is measured using a Brookfield LVDV-II+Programmable Viscometer at 25° C., or an equivalent such as BrookfieldDV II+, Spindle LV2 at 60 or 100 rpm, depending on the viscosity. Forthe programmable viscometer, the procedure used was based on theOperating Instructions, Manual No. M/97-164. This Viscometer willdetermine viscosity only if the correct spindle and rpm is used for theviscosity of the sample according to instruction manual.

[0050] It is preferable that the properties of a creping agent areapproximately the same subsequent to treatment as they were prior totreatment. Therefore, as noted above, preferably, the viscosity of thereaction mixture is maintained constant or substantially constant duringthe reaction for creping agents. In particular, the viscosity of thereaction mixture does not increase more than about 50%, more preferablyno more than about 20%, and most preferably no more than about 10% fromthe starting viscosity.

[0051] It is further noted that conditions, preferably temperature, pHand concentration of enzymatic agent, can be varied during the reaction.For example, if the viscosity of the reaction mixture is higher thandesired, the pH and/or temperature can be lowered and/or additionalenzymatic agent can be added. Conversely, for example, if the viscosityof the reaction mixture is lower than desired, the pH and/or temperaturecan be raised.

[0052] The present invention is also directed to a process of reducingmolecular weight or viscosity of a polyamine-epihalohydrin resincontaining composition, comprising treating the composition containingpolyamine-epihalohydrin resin with at least one enzymatic agent. Thecomposition can comprise a high solids contents, such as a solidscontent of at least 15 wt %. Varying of the reaction conditions usuallywill change the time of the reaction. The pH and/or temperature can belowered and/or additional enzymatic agent can be added.

[0053] For wet-strength resins and with using ALCALASE 2.5L type DX asthe enzyme, specific examples of preferable conditions include thefollowing. With a resin having a starting Brookfield viscosity of about150 to 300 cps and about 20-22 wt % active solids, it is preferred touse a temperature of about 20-33° C., a pH of about 6.8-7.8, an ALCALASE2.5L type DX (as received basis) to active solids ratio of about 1.0:20to 1.0:5.0. More specifically, with Kymene® E7219 (available fromHercules Incorporated, Wilmington, Del.) having a starting Brookfieldviscosity about 200 to 300 cps with about 20-22% active solids, it ispreferred to use a temperature of about 23-27° C., a pH of about6.8-7.5, an ALCALASE 2.5L type DX (as received basis) to active solidsratio of about 1.0:8.0 to 1.0:18.0 with a treatment time of 6-10 hours.It should be noted that as the treatment time is increased, the amountof CPD released from the CPD-producing species is desirably increased,with a preferred treatment time being 6 to 10 hours. Another example ofconditions is the following: Kymene® E7219 (available from HerculesIncorporated., Wilmington, Del.) having a starting Brookfield viscosityabout 200 to 300 cps with about 20-22% active solids, a temperature ofabout 35° C., a pH of about 7.5, an ALCALASE 2.5L type DX (as receivedbasis) weight to active solids ratio(weight) of about 1.0:8.3.

[0054] The temperature can be at least about 0° C., more preferablyabout 10° C. to 80° C., even more preferably about 20° C. to 60° C.,more preferably about 20° C. to 40° C. and more preferably about 20° C.to 30° C. The reaction time can be about 3 minutes to 350 hours, morepreferably about 30 minutes to 48 hours, more preferably about 30minutes to 96 hours, more preferably about 1 hour to 24 hours, and evenmore preferably about 2 hours to 12 hours. The pH of the enzymatictreatment will depend on the pH dependence of the specific enzyme andthe other treatment conditions, and can vary between 1 to 11, preferably2 to 10, even more preferably about 2.5 to 9, and even more preferablyabout 7-9, and even more preferably 7 to 8. Additional preferred pHranges include; 5.0 to 8.0, 5.5to 7.5, 6 to 9, 6 to 8.5, 6.5 to 8.

[0055] For example, the combined treatment can be started at pH 6.8-7.8for the first 4-24 hours and than lowered to pH of 5.5-7.0 or the pH canbe allowed to drift down to 6.5-7.2 for the latter 8-48 hours of thecombined treatment.

[0056] The concentration of the enzyme will depend upon its activity.For example, but not limited to, the enzyme can be present in an amountof about 0.04 g of active enzyme (dry basis) to 1600 gpolyamine-epichlorohydrin resin (dry basis) to 0.04 g of active enzyme(dry basis) to 1.5 g polyamine-epichlorohydrin resin (dry basis), alsothe enzyme can be present in an amount of about 0.04 g of active enzyme(dry basis) to 160 g polyamine-epichlorohydrin resin (dry basis) to 0.04g of active enzyme (dry basis) to 4 g polyamine-epichlorohydrin resin(dry basis).

[0057] The concentration of the enzyme will depend upon its activity.For example, but not limited to, in the case of ALCALASE, the enzyme canbe present in an amount of about 1 g of ALCALASE 2.5L type DX (asreceived) to 1600 g polyamine-epichlorohydrin resin (dry basis) to 1 gof ALCALASE 2.5L type DX (as received) to 1.5 gpolyamine-epichlorohydrin resin (dry basis), also the enzyme can bepresent in an amount of about 1 g of ALCALASE 2.5L type DX (as received)to 160 g polyamine-epichlorohydrin resin (dry basis) to 1 g of ALCALASE2.5L type DX (as received) to 4 g polyamine-epichlorohydrin resin (drybasis).

[0058] It is noted that following the guidelines and the non-limitingexamples, set forth in the instant application one having ordinary skillin the art would be capable of determining treatment conditions and thebalancing of treatment conditions to obtain hydrolysis of CPD-formingspecies at high solids concentrations and/or to obtain a reduction inmolecular weight or viscosity. For example, as the solids concentrationincreases, the pH and/or temperature should usually be decreased, andthe enzymatic agent concentration will usually be increased. Moreover,following the guidelines, one having ordinary skill in the art would becapable of determining enzymatic agents that are useful to removeCPD-forming species and/or to obtain a reduction in molecular weight orviscosity.

[0059] Moreover, preferred reaction conditions can be varied by usingappropriate types and amounts of enzymes. For example, if the enzymaticagent has higher protease as compared to esterase activity (protease/esterase balance) with a polyamine-epichlorohydrin resin, then reactionconditions could be varied to higher pH, temperature and/or solids, suchas reaction conditions above about pH 8 and/or temperature above about40° C. and/or solids as high as about 40 wt %. Practical being definedas obtaining a reduced CPD-forming resin while having a resin with thedesired viscosity. Although conditions will be dependent on the balanceof esterase and protease activity of a particular enzyme, the preferredconditions with the present invention with ALCALASE 2.5 L type DX arethe following: 15-50 wt % active solids, pH 6.9 to 7.9, at 0 to 35° C.,for 4 to 24 hours and 8-20 g of active solids for 1 g of ALCALASE 2.5 Ltype DX (as received), and starting viscosity of 10 cP to 1000 cP.Moreover, it is noted that throughout the application the terminologyenzymatic agent concentration is utilized. However, one having ordinaryskill in the art would understand that enzymes can have differentactivities, and the concentration of the enzyme can be adjusteddepending upon the activity.

[0060] The enzyme treatment can be applied on resins as produced in aresin synthesis process without further treatment. Moreover, the resinscan be treated by various processes prior to reduction and/or removal ofthe CPD-forming species. Still further, after treatment to reduce and/orremove CPD-forming species, the resin can be treated by variousprocesses. Yet still further, the resin can be treated by variousprocesses prior to reduction and/or removal of the CPD-forming species,and the resin can also be treated by various processes after treatmentto reduce and/or remove CPD-forming species. For the sake of brevity, acomplete description of these processes is not being repeated herein,and reference is made to the above-identified U.S. patent applicationSer. Nos. 09/629,629, 09/592,681, 09/363,224 and 09/330,200, which areincorporated by reference herein in their entireties.

[0061] The resins according to the present invention are capable ofbeing stored without undue formation of CPD. More specifically, as anexample, the solution will contain less than about 10 ppm (parts permillion), more preferably less than about 5 ppm, and most preferablyless than 1 ppm of CPD, when stored at about 13.5 wt % resin solidscontent. In the context of the present invention the phrase “resinsolids” means the active polyamine-epihalohydrin of the composition.

[0062] To determine storage stability of resin solutions according tothe present invention, a resin solution stability test is performedwherein the resin solution is stored for a period of 2 weeks at 50° C.,and a pH of about 2.5 to 8, preferably 2.8, and the CPD content ismeasured at the end of the 2 week period. Thus, a solution containingpolyamine-epihalohydrin resin according to the present invention will bestorage stable if it contains less than about 250 ppm dry basis of CPDwhen measured at the end of the two week period, more preferably lessthan about 150 ppm dry basis of CPD when measured at the end of the 2week period, more preferably less than about 75 ppm dry basis of CPDwhen measured at the end of the 2 week period, even more preferably lessthan about 40 ppm dry basis of CPD when measured at the end of the twoweek period, and even more preferably less than about 10 ppm dry basisof CPD when measured at the end of the 2 week period.

[0063] The resin solution stability test can be performed on solutionscontaining varying percent resin solids content; however, the CPDproduced should be corrected for solids content. For example, for a 15wt % resin solids content solution having a measured CPD content of 15ppm, the corrected CPD, on a dry basis, will be 100 ppm dry basis (15ppm/0.15 weight resin solids content).

[0064] The resin solution stability test is performed by charging aportion of the polyamine-epihalohydrin resin into a container containinga stirrer. The container is placed in a 50° C. water bath and maintainedat 50° C. with stirring. An aliquot is removed from the container andsubmitted for GC (gas chromatography) analysis according to the GCprocedure as set forth below. Typically, a flame ionization detector(FID) is first used to analyze the sample. An electrolytic conductivitydetector (ELCD) or a halogen-specific detector (XSD) is used whenincreased sensitivity is needed, especially at less than about 20 ppm ofthe species to be analyzed. Other sensitive detectors can be used, e.g.,electron capture detectors. This test is an accelerated aging test tomodel aging at longer periods of time at about 32° C.

[0065] An additional test to determine storage stability of resinsolutions according to the present invention is the following test(“Acid Test”): A portion of resin to be tested is charged into acontainer containing a stirrer. The pH is adjusted to 1.0 with 96 wt %sulfuric acid. The container is closed and placed in a 50° C. water bathand maintained at 50° C. with stirring. An aliquot is removed from thecontainer at 24 hours, and submitted for GC analysis in the mannerdescribed below to provide an indication of the storage stability.

[0066] The acid test can be performed on solutions containing varyingpercent resin solids content; however, the CPD produced should becorrected for solids content. For example, for a 15 wt % resin solidscontent solution having a measured CPD content of 15 ppm, the correctedCPD, on a dry basis, will be 100 ppm dry basis (15 ppm/0.15 weight resinsolids content).

[0067] For the embodiment of the invention where the enzyme treatment isapplied to the resins in a resin synthesis process without need forfurther treatment, although further treatment can be used, the amount ofCPD release and/or produced by the resin, when stored at pH 1 for 24hours at 50° C. and measured at 24 hours, releases and/or produces lessthan about 1000 ppm dry basis of CPD, more preferably releases and/orproduces less than about 750 ppm dry basis of CPD, even more preferablyreleases and/or produces less than about 500 ppm dry basis of CPD, evenmore preferably releases and/or produces less than about 250 ppm drybasis of CPD, even more preferably releases and/or produces less thanabout 200 ppm dry basis of CPD, even more preferably releases and/orproduces less than about 150 ppm dry basis of CPD, even more preferablyreleases and/or produces less than about 100 ppm dry basis of CPD, evenmore preferably releases and/or produces less than about 75 ppm drybasis of CPD, even more preferably releases and/or produces less thanabout 50 ppm dry basis of CPD, even more preferably releases and/orproduces less than about 25 ppm dry basis of CPD, even more preferablyreleases and/or produces less than about 15 ppm dry basis of CPD, evenmore preferably releases and/or produces less than about 5 ppm dry basisof CPD, and even more preferably releases and/or produces less thanabout 3 ppm dry basis of CPD, and even more preferably releases and/orproduces less than about 1 ppm dry basis of CPD.

[0068] For the embodiment of the invention where the enzyme treatment issimultaneously with, prior to or subsequent to an additional treatmentto reduce at least one of epihalohydrins, epihalohydrin byproducts andorganic halogen bound to the polymer backbone this additional treatmentcan be, but is not limited to, contacting the reduced CPD-forming resinwith at least one microorganism, or at least one enzyme isolated fromthe at least one microorganism, in an amount, and at a pH andtemperature effective to dehalogenate residual quantities of organicallybound halogen, when stored at pH 1 for 24 hours at 50° C. and measuredat 24 hours, contains less than about 1000 ppm dry basis of CPD, morepreferably contains less than about 750 ppm dry basis of CPD, even morecontains less than about 500 ppm dry basis of CPD, even more preferablycontains less than about 250 ppm dry basis of CPD, even more preferablycontains less than about 200 ppm dry basis of CPD, even more preferablycontains less than about 150 ppm dry basis of CPD, even more preferablycontains less than about 100 ppm dry basis of CPD, even more preferablycontains less than about 75 ppm dry basis of CPD, even more preferablycontains less than about 50 ppm dry basis of CPD, even more preferablycontains less than about 25 ppm dry basis of CPD, even more containsless than about 15 ppm dry basis of CPD, even more preferably containsless than about 5 ppm dry basis of CPD, and even more preferablycontains less than about 3 ppm dry basis of CPD, and even morepreferably contains less than about 1 ppm dry basis of CPD.

[0069] GC Procedure and Instrumentation: GC was used to determine epiand epi by-products in the treated and untreated resins using thefollowing method. The resin sample was absorbed onto an Extrelut column(available from EM Science, Extrelut QE, Part #901003-1) and extractedby passing ethyl acetate through the column. A portion of the ethylacetate solution was chromatographed on a wide-bore capillary column. Ifflame ionization detector (FID) was used, the components are quantitatedusing n-octanol as the internal standard. If an electrolyticconductivity (ELCD) detector or if the halogen-specific (XSD) detectorwas used, an external standard method using peak matching quantitationwas employed. The data system was either a Millennium 2010 or HPChemStation. The FID detector was purchased from Hewlett-Packard (HP) aspart of a Model 5890 GC. The ELCD detector, Model 5220, was purchasedfrom OI Analytical. The XSD detector was purchased from OI Analytical,Model 5360 XSD. The GC instrument used was a HP Model 5890 series II.The column was DB-WAX (Megabore, J&W Scientific, Inc.) 30 m×0.53 mm with1.5 micron film thickness. For the FID and ELCD, the carrier gas washelium with a flow rate of 10 mL/min. The oven program was 35° C. for 7minutes, followed by ramping at 8° C./min to 200° C. and holding at 200°C. for minutes. The FID used hydrogen at 30 mL/min and air at 400 mL/minat 250° C. The ELCD used n-propanol as the electrolyte with anelectrolyte flow rate setting of 50% with a reactor temperature of 900°C. The XSD reactor was operated in an oxidative mode at 1100° C. with ahigh purity air flow rate of 25 mL/min.

[0070] Moreover, paper products containing resins according to thepresent invention are capable of being stored without undue formation ofCPD. Thus, paper products according to the present invention can haveinitial low levels of CPD, and can maintain low levels of CPD over anextended period storage time. More specifically, paper productsaccording to the present invention, made with a 1 wt % addition level ofresin, will contain less than about 250 parts per billion (ppb) of CPD,more preferably less than about 100 ppb of CPD, even more preferablyless than about 50 ppb of CPD and even more preferably less than about10 ppb of CPD, and even more preferably less than about 1 ppb of CPDwhen stored for periods as long as 2 weeks, preferably as long as atleast 6 months, and even more preferably as long as at least one year.Moreover, paper products according to the present invention, made withabout a 1 wt % addition level of resin, will have an increase in CPDcontent of less than about 250 ppb, more preferably less than about 100ppb of CPD, even more preferably less than about 50 ppb of CPD, evenmore preferably less than about 10 ppb of CPD, and even more preferablyless than about 1 ppb of CPD when stored for periods as long as 2 weeks,more preferably as long as at least 6 months, and even more preferablyas long as at least one year. In other words, the paper productsaccording to the present invention have storage stability and will notgenerate excessive CPD content in paper products when the paper productsare stored as little as one day and for periods of time greater than oneyear. Thus, the resins according to the present invention give minimalformation of CPD in paper products, particularly those exposed toaqueous environments, especially hot aqueous environments, e.g., teabag, coffee filters, etc. Further examples of paper products includepackaging board grade, and tissue and towel grade.

[0071] Paper can be made by adding the resin at addition levels otherthan about 1 wt %; however, the CPD content should be corrected for theaddition level. For example, for a paper product made by adding theresin at a 0.5 wt % addition level having a measured CPD content of 50ppb, the corrected CPD on a 1 wt % addition level basis will be 100 ppb(50 ppb/0.5 percent addition level).

[0072] To measure CPD in paper products, the paper product is extractedwith water according to the method described in European standard EN647, dated October 1993. Then 5.80 grams of sodium chloride is dissolvedinto 20 ml of the water extract. The salted aqueous extract istransferred to a 20 gram capacity Extrelut column and allowed tosaturate the column for 15 minutes. After three washes of 3 ml ethylacetate and saturation of the column, the Extrelut column is eluteduntil 300 ml of eluent has been recovered in about 1 hour. The 300 ml ofethyl acetate extract is concentrated to about 5 ml using a 500-mlKuderna-Danish concentrating apparatus (if necessary, furtherconcentrating is done by using a micro Kudema-Danish apparatus). Theconcentrated extract is analyzed by GC using the procedure andinstrumentation described above. Typically, an electrolytic conductivitydetector (ELCD) or a halogen-specific detector (XSD) is used. Othersensitive detectors can be used, e.g., electron capture detectors.Alternatively, CPD in paper products can be measured using the proceduredescribed in Example 4.

[0073] The resins that can be treated with enzymatic agent according tothe present invention can comprise any polyamine-epihalohydrin resins.This invention is also directed towards the preparation, use andtreatment of polyamine-epihalohydrin resins, such aspolyaminopolyamide-epichlorohydrin resins, made by reactingepihalohydrin, such as epichlorohydrin, with a prepolymer (alsointerchangeably referred to herein as polymer), such as polyaminoamideprepolymer. In the case of polyaminopolyamide resins, it is noted thatthe polyaminoamide prepolymer is also referred to as polyamidoamine,polyaminopolyamide, polyamidopolyamine, polyamidepolyamine, polyamide,basic polyamide, cationic polyamide, aminopolyamide, amidopolyamine orpolyaminamide.

[0074] A preferred group of polymers for use in the present inventionincludes cationic polymers, alone or together with other polymers.Particularly preferred cationic polymers include those used for thepurpose of imparting wet strength to paper as well as creping agents. Alisting of many polymers useful in papermaking formulations, such as wetstrength and creping agents, is described in Paper Chemistry, ISBN0-216-92909-1, pages 78-96, published in the USA by Chapman Hall, NewYork. Chapter 6 of this book is entitled “Wet Strength Chemistry”, andis hereby incorporated, in its entirety, by reference thereto. Chapter 6describes several classes of polymers which are used to impart wetstrength to paper, including: polyaminoamide-epichlorohydrin resin,urea-formaldehyde resin, melamine-formaldehyde resin, epoxidizedpolyamide resin, glyoxalated polyacrylamide resin, polyethyleneimineresin, dialdehyde starch, proteinaceous adhesive treated withformaldehyde, cellulose xanthate (viscose), synthetic latex, vegetablegum, and glyoxal. The polyaminoamide-epichlorohydrin resin may be aKymene® brand polyaminoamide-epichlorohydrin resin, such as Kymene®557LX, Kymene® SLX2, or Kymene® 617, or a polyamine-epichlorohydrinresin such as Kymene® 2064, Kymene® 367 resins, and Kymene® 736 orpolyamide-polyurylene-epihalohydrin resins such as Kymene® 450.

[0075] The invention is directed to cationic polymers such aspolyamine-epichlorohydrin resins which may be used alone or incombination with other polymers used for the wet strengthening of paperand creping agents. These resins include epichlorohydrin resins andnitrogen-containing cationic polymers, both of which are derived fromepichlorohydrin reactants. Preferred resins for the purposes of thisinvention include polyaminoamide-epichlorohydrin wet-strength resins asdescribed in U.S. Pat. Nos. 2,926,154; 3,332,901; 3,891,589; 3,197,427;4,240,935, 4,857,586; European Patent Publication 0,349,935, and GreatBritain Patent 865,727, and U.S. patent application Ser. Nos.09/629,629, 09/592,681, 09/363,224 and 09/330,200. Further, resinsinclude Crepetrol® 80E or Crepetrol® A3025, Crepetrol® A6115, Crepetrol®A8225, Crepetrol® 870, SPC 003, and Rezosol® 8289 creping agents, whichare available from Hercules Incorporated, Wilmington, Del. It is notedthat these resins are generally referred to herein aspolyamine-epihalohydrin resins, and such resins include, but are notlimited to, polyaminopolyamide-epihalohydrin resins (which are alsoknown as polyaminoamide-epihalohydrin resins,polyamidepolyamine-epihalohydrin resins,polyaminepolyamide-epihalohydrin resins, aminopolyamide-epihalohydrinresins, polyamide-epihalohydrin resins); polyalkylenepolyamine-epihalohydrin; and polyaminourylene-epihalohydrin resins,copolyamide-polyurylene-epihalohydrin resins,polyamide-polyurylene-epihalohydrin resins, with the epihalohydrinpreferably being epichlorohydrin in each instance. Processes for makingthese known resins are also disclosed in these documents, which areincorporated in their entireties, by reference thereto.

[0076] Exemplary epichlorohydrin resins in these patents arecharacterized by the presence of N-chlorohydrin groups of the formula:

[0077] and quaternary N-chlorohydrin groups of the formula:

[0078] Wherein the tetrasubstituted nitrogen atom is positively charged(a quaternary nitrogen), and hence cationic; and isomeric3-hydroxyazetidinium chloride groups of the formula:

[0079] A preferred cationic polymer utilized in the present invention isa polymer having the following formula:

[0080] where the asterisked tetrasubstituted nitrogen atom is positivelycharged (a quaternary nitrogen), and hence cationic. The nitrogen atomis in a 4-membered ring (i.e. a 3-hydroxyazetidinium group). Otheruncharged polymer units also co-exist along polymer chains of this typeof resin. Even though a few negatively charged (i.e., anionic) groupsmay also be present on the polymer, the net charge along the polymerchain is positive. X⁻ is a simple anion, which is not covalently bondedto the polymer chain. Generally the anion is a chloride ion, and n is aninteger of from about 5 to several thousand, preferably 5 to 3000.

[0081] Creping agents include, without limitation, Crepetrol® 80E orCrepetrol® A3025, Crepetrol® A6115, Crepetrol® A8225, Crepetrol® 870,SPC 003, and Rezosol® 8289 creping agents.

[0082] For wet strength agents, while ratios greater than 1 can beutilized, it is preferred that the resin comprise a resin formed in apolyamide-epihalohydrin reaction having a molar ratio of epihalohydrinto secondary amine group of less than 1, more preferably the molar ratioof epihalohydrin to secondary amine group is less than about 0.975, witha preferred range of the molar ratio of epihalohydrin to secondary aminegroup being about 0.5 to 0.975, more preferably the molar ratio ofepihalohydrin to secondary amine group being about 0.6 to 0.975, andeven more preferably about 0.8 to 0.975. For creping agents, it ispreferred that the resin comprise a resin formed in apolyamide-epihalohydrin reaction having a molar ratio of epihalohydrinto secondary amine group of less than about 0.50, more preferably lessthan about 0.25, and can even be lower than 0.1, with a preferred lowerlimit of about 0.05.

[0083] Moreover, creping agents according to the present invention donot need as much crosslinking functionalities as wet strength agents,and can therefore have a lower azetidinium level than wet strengthagents. Thus, preferably the azetidinium level of creping agents is lessthan about 10 mole %, with a preferred range of about 5 to 10 mole %,and preferably the azetidinium level of wet strength agents is greaterthan about 30 mole %, with a preferred range of about 30 to 70 mole %.The mole % azetidinium and the mole % of other species can be determinedby the following NMR Procedure. NMR Procedure:

[0084] The ¹³C NMR spectra are acquired using BRUKER AMX spectrometersequipped with a 10 mm broadband probe. A ¹³C NMR operating frequency of100 MHz (AMX400) or 125 MHz (AMX500) is sufficient for data collection.In either case, the spectra are acquired with continuous ¹H decoupling.Electronic integration of the appropriate signals provides molarconcentrations of the following alkylation components; ACH, EPX, GLY,and AZE.

[0085] where: ACH=polymeric aminochlorohydrins, EPX=polymeric epoxides,GLY=polymeric glycols, AZE=azetidinium ions

[0086] In order to calculate the concentrations of each of thesespecies, the integral values must be placed on a one (1) carbon basis.For example, the spectral region between 20-42 ppm represents six (6)carbons of the diethylenetriamine-adipate backbone, hence the integralvalue is divided by six. This value is used as the polymer commondenominator (PCD) for calculation of the alkylation species. Thechemical shifts of these species are provided below (using anacetonitrile field reference of 1.3 ppm). The corresponding integralvalue of each alkylation product is used in the numerator forcalculation, refer to examples below:

[0087] ACH signal at 68-69 ppm represents one carbon; integral ofACH÷PCD=mole fraction ACH

[0088] GLY signal at 69-70 ppm represents one carbon; integral ofGLY÷PCD=mole fraction GLY

[0089] EPX carbon at 51-52ppm represents one carbon; integral ofEPX÷PCD=mole fraction EPX

[0090] AZE signal at 73-74 ppm represents two carbons, thus, a divisionfactor of two is required; integral of AZE/2÷PCD=mole fraction AZE

[0091] The following spectral parameters are standard experimentalconditions for ¹³C NMR analysis of Kymene resins or creping agents onthe Bruker AMX400. Temperature 25 C. Resonance Frequency 100 MHz # DataPoints 64K Dwell Time 20 microseconds Acquisition Time 1.3 seconds SweepWidth 25000 Hz Number of Scans  1K Relaxation Delay   3 seconds PulseTip Angle 70 degrees Pulse Program zgdc Processed Spectral Size 64KApodization Function exponential Line Broadening   3 Hz

[0092] Moreover according to the present invention, for creping agentsderived from prepolymers containing tertiary amine functionality, thecreping agent will preferably have a quaternary aminohalohydrin, e.g.,aminochlorohydrin, content of less than about 30 mole %, while wetstrength agents according to the present invention preferably have aquaternary aminohalohydrin, e.g., aminochlorohydrin, content of greaterthan 30 mole %. Moreover, without wishing to be bound by theory, it isbelieved that secondary amine compounds, such as diethylenetriamine,form azetidinium groups, whereas, tertiary amine type compounds, such asmethylbis(3-aminopropyl)amine, form quaternary aminochlorohydrin groups.Examples of tertiary amine type compounds include, but are not limited,the reaction product of adipic acid and a methylbis(3-aminopropyl)amine,result in a tertiary amine prepolymer. This prepolymer is used to make atertiary amine based resin which contains quaternary aminohalohydringroups.

[0093] Preferred polyamines for this invention are produced by reactinga dicarboxylic acid, or a derivative thereof, with methylbis(3-aminopropyl)amine or with a polyalkylenepolyamine containing fromtwo to four alkylene groups having two to four carbons, two primaryamine groups, and one to three secondary amine groups. Dicarboxylic acidderivatives suitable for preparing the polyaminoamides include esters,anhydrides and acid halides.

[0094] Procedures for preparing polyaminoamides frompolyalkylenepolyamines are described in U.S. Pat. No. 2,926,154, toKeim, which is incorporated herein by reference in its entirety.Procedures utilizing methyl bis(3-aminopropyl)amine for preparation ofpolyaminoamides are described in U.S. Pat. No. 5,338,807 to Espy et al.and U.S. Pat. No. 5,994,449, which are incorporated by reference hereinin their entireties.

[0095] Expanding upon the above, polyaminopolyamide-epichlorohydrinresins comprise the water-soluble polymeric reaction product ofepichlorohydrin and polyamide derived from polyalkylene polyamine andsaturated aliphatic dibasic carboxylic acid containing from about 2 toabout 10 carbon atoms. It has been found that resins of this type impartwet-strength to paper whether made under acidic, alkaline or neutralconditions. Moreover, such resins are substantive to cellulosic fibersso that they may be economically applied thereto while the fibers are indilute aqueous suspensions of the consistency used in paper mills.

[0096] In the preparation of the cationic resins contemplated for useherein, the dibasic carboxylic acid is first reacted with thepolyalkylene polyamine, under conditions such as to produce awater-soluble polyamide containing the recurring groups

—NH(C_(n)H_(2n)NH)_(x)—CORCO—

[0097] where n and x are each 2 or more and R is the divalenthydrocarbon radical of the dibasic carboxylic acid. This water solublepolyamide is then reacted with an epihalohydrin to form thewater-soluble cationic resins. The dicarboxylic acids contemplated foruse in preparing the resins of the invention are the saturated aliphaticdibasic carboxylic acids containing from 2 to 10 carbon atoms such asoxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid,azelaic acid and the like. The saturated dibasic acids having from 4 to8 carbon atoms in the molecule, such as adipic and glutaric acids arepreferred. Blends of two or more of the saturated dibasic carboxylicacids may also be used. Derivatives of dibasic carboxylic acids, such asesters, half-esters and anhydrides can also be used in the presentinvention, such as dimethyl adipate, diethyl adipate, dimethylglutarate, diethyl glutarate, dimethyl succinate and diethyl succinate.Blends of two or more of derivatives of dibasic carboxylic acids mayalso be used, as well as blends of one or more derivatives of dibasiccarboxylic acids with dibasic carboxylic acids.

[0098] A variety of polyalkylene polyamines including polyethylenepolyamines, polypropylene polyamines, polybutylene polyamines,polypentylene polyamines, polyhexylene polyamines and so on and theirmixtures may be employed of which the polyethylene polyamines representan economically preferred class. More specifically, the polyalkylenepolyamines contemplated for use may be represented as polyamines inwhich the nitrogen atoms are linked together by groups of the formula—C_(n)H_(2n)— where n is a small integer greater than unity and thenumber of such groups in the molecule ranges from two up to about eight.The nitrogen atoms may be attached to adjacent carbon atoms in the group—C_(n)H_(2n)— or to carbon atoms further apart, but not to the samecarbon atom. This invention contemplates not only the use of suchpolyamines as diethylenetriamine, triethylenetetramine,tetraethylenepentamine and dipropylenetriamine, which can be obtained inreasonably pure form, but also mixtures and various crude polyaminematerials. For example, the mixture of polyethylene polyamines obtainedby the reaction of ammonia and ethylene dichloride, refined only to theextent of removal of chlorides, water, excess ammonia, andethylenediamine, is a satisfactory starting material. The term“polyalkylene polyamine” employed in the claims, therefore, refers toand includes any of the polyalkylene polyamines referred to above or toa mixture of such polyalkylene polyamines and derivatives thereof.Additional polyamines that are suitable for the present inventioninclude; bis-hexamethylenetriamine (BHMT), methylbisaminopropylamine(MBAPA), other polyalkylene polyamines (e.g., spermine, spermidine).Preferably, the polyamines are diethylenetriamine, triethylenetetramine,tetraethylenepentamine and dipropylenetriamine.

[0099] It is desirable, in some cases, to increase the spacing ofsecondary amino groups on the polyamide molecule in order to change thereactivity of the polyamide-epichlorohydrin complex. This can beaccomplished by substituting a diamine such as ethylenediamine,propylenediamine, hexamethylenediamine and the like for a portion of thepolyalkylene polyamine. For this purpose, up to about 80% of thepolyalkylene polyamine may be replaced by molecularly equivalent amountof the diamine. Usually, a replacement of about 50% or less will servethe purpose.

[0100] Appropriate aminocarboxylic acids containing at least threecarbon atoms or lactams thereof are also suitable for use to increasespacing in the present invention. For example, 6-aminohexanoic acid andcaprolactam.

[0101] Polyaminoureylene-epihalohydrin resins, particularlypolyaminoureylene-epichlorohydrin resins, are also contemplated in thepresent invention, such as discussed in U.S. Pat. Nos. 4,487,884 and3,311,594, which are incorporated by reference in their entireties, suchas Kymene®450 type of resins (Hercules Incorporated, Wilmington, Del.).The polyaminoureylene resins contemplated for preparation and use hereinare prepared by reacting epichlorohydrin with polyaminoureylenescontaining free amine groups. These polyaminoureylenes are water-solublematerials containing tertiary amine groups and/or mixtures of tertiaryamine groups with primary and/or secondary amino groups and/orquaternary ammonium groups. However, tertiary amino groups shouldaccount for at least 70% of the basic nitrogen groups present in thepolyaminoureylene. These polyaminoureylenes may be prepared by reactingurea or thiourea with a polyamine containing at least three aminogroups, at least one of which is a tertiary amino group. The reactioncan, if desired, be carried out in a suitable solvent such as xylene.

[0102] The polyamine reactant should preferably have at least threeamino groups, at least one of which is a tertiary amino group. Thepolyamine reactant may also have secondary amino groups in limitedamounts. Typical polyamines of this type suitable for use as hereinabovedescribed are methyl bis(3-aminopropyl)amine (MBAPA), methylbis(2-aminoethyl)amine, N-(2-aminoethyl)piperazine,4,7-dimethyltriethylenetetramine and so on, which can be obtained inreasonably pure form, but also mixtures of various crude polyaminematerials.

[0103] To prepare the prepolymer from diacid and polyalkylenepolyamine,a mixture of the reactants is preferably heated at a temperature ofabout 125-200° C. for preferably about 0.5 to 4 hours, at atmosphericpressure. Where a reduced pressure is employed, lower temperatures suchas 75° C. to 150° C. may be utilized. This polycondensation reactionproduces water as a byproduct, which is removed by distillation. At theend of this reaction, the resulting product is dissolved in water at aconcentration of about 50% by weight total polymer solids.

[0104] Where diester is used instead of diacid, the prepolymerizationcan be conducted at a lower temperature, preferably about 100-175° C. atatmospheric pressure. In this case the byproduct will be an alcohol, thetype of alcohol depending upon the identity of the diester. Forinstance, where a dimethyl ester is employed the alcohol byproduct willbe methanol, while ethanol will be the byproduct obtained from a diethylester. Where a reduced pressure is employed, lower temperatures such as75° C. to 150° C. may be utilized.

[0105] In converting the polyamide, formed as above described, to acationic resin, it is reacted with epichlorohydrin at a temperature fromabove about 0° C., more preferably about 25° C., to about 100° C., andpreferably between about 35° C. to about 70° C. until the viscosity of a20% solids solution at 25° C. has reached about C or higher on theGardner Holdt scale. This reaction is preferably carried out in aqueoussolution to moderate the reaction. Although not necessary, pH adjustmentcan be done to increase or decrease the rate of crosslinking.

[0106] When the desired viscosity is reached, sufficient water can beadded to adjust the solids content of the resin solution to the desiredamount, i.e., about 15 wt % more or less, the product can be cooled toabout 25° C. and then stabilized to permit storage by improving thegelation stability by adding sufficient acid to reduce the pH to lessthan about 6, preferably less than about 5, and most preferably lessthan about 4. Any suitable inorganic or organic acid such ashydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid,formic acid, phosphoric acid and acetic acid may be used to stabilizethe product. Non-halogen containing acids, such as sulfuric acid, arepreferred.

[0107] Fibrous webs are creped using the compositions of this inventionby: (1) applying the composition described above to a drying surface forthe web or to the web; (2) pressing the fibrous web against the dryingsurface to effect adhesion of the web to the drying surface; and (3)dislodging the web from the drying surfaces with a creping device suchas a doctor blade to crepe the fibrous web. Preferably, in step (1), thecomposition is applied to the drying surface for the web. The preferredfibrous web is a cellulosic web.

[0108] Preferably the creping adhesive is applied in aqueous solutioncontaining from about 0.1 to about 10 weight percent of the resincomposition. More preferably, the resin composition is in solution atthe level of about 0.25 to about 5 weight percent, and most preferablyat about 0.5 to about 2 weight percent. For creping agents on a dryweight basis, a minimum amount of about 0.001 weight percent based onthe dry weight of the pulp or paper is used. A more preferable minimumamount is about 0.005 weight percent, and the most preferable minimumamount is about 0.01 weight percent. The preferable maximum amount ofresin composition is about 2 weight percent. A more preferable maximumis about 1 weight percent, and the most preferable maximum about 0.5weight percent. The drying surface most commonly used in commercialoperations is a Yankee dryer, and the aqueous solution of adhesive willmost often be applied to the creping cylinder or drum by spraying.Alternatively, however, it can be added by application to the fibrousweb, preferably by spraying. In the case of cellulose webs, i.e. paper,the creping adhesive can be added at the wet end of the paper machine byapplication to the wet web. In some situations it may be possible to addthe creping adhesive to the pulp before formation of the sheet.

[0109] Other ingredients, in particular, agents which modify adhesion ofthe web to the drying surface, can used in conjunction with the crepingadhesives of this invention. Such agents, also know as release agents orplasticizers, include water soluble polyols, glycols, polyethyleneglycols, sugars, oligosaccharides and hydrocarbon oils.

[0110] The process for making paper utilizing the resin compositions ofthis invention comprises: (a) providing an aqueous pulp suspension; (b)adding to the aqueous pulp suspension the resin and (c) sheeting anddrying the aqueous pulp suspension produced in (b) to obtain paper.

[0111] The aqueous pulp suspension of step (a) of the process isobtained by means well known in the art, such as known mechanical,chemical and semichemical, etc., pulping processes. Normally, after themechanical grinding and/or chemical pulping step, the pulp is washed toremove residual pulping chemicals and solubilized wood components.Either bleached or unbleached pulp fiber may be utilized in the processof this invention. Recycled pulp fibers are also suitable for use.

[0112] In step (b), resin of this invention preferably is added to pulpslurry in a minimum amount of about 0.1 weight percent based on the dryweight of the pulp. A more preferable minimum amount is about 0.2 weightpercent. The preferable maximum amount of resin composition is about 5weight percent. A more preferable maximum is about 3 weight percent, andthe most preferable maximum about 1.5 weight percent. The resincomposition is generally added in the form of an aqueous solution. Inaddition to the resin, other materials normally used in paper may beadded as well. These include, for example, sizing agents, pigments,alum, brightening agents, dyes and dry strength agents, added in amountswell known in the art.

[0113] Step (c) is carried out according to procedures well known tothose skilled in the art of papermaking.

[0114] As discussed above, resins having at least reduced levels offormation of CPD can be resins as produced in a resin synthesis processwithout further treatment. Moreover, the resins can be treated byvarious processes prior to reduction and/or removal of the CPD-formingspecies. Still further, after treatment to reduce and/or removeCPD-forming species, the resin can be treated by various processes. Yetstill further, the resin can be treated by various processes prior toreduction and/or removal of the CPD-forming species, and the resin canalso be treated by various processes after treatment to reduce and/orremove CPD-forming species. For example, the resin can be treated byvarious processes, such as processes to remove low molecular weightepihalohydrin and epihalohydrin by-products, e.g., epichlorohydrin andepichlorohydrin by-products, for example, CPD in the resin solution.Without limiting the treatments or resins that can be utilized, it isnoted that resins, such as Kymene®SLX2, Kymene®617 and Kymene®557LX(available from Hercules Incorporated, Wilmington, Del.), and Crepetrol®80E or Crepetrol® A3025, Crepetrol® A6115, Crepetrol® A8225, Crepetrol®870, SPC 003, and Rezosol® 8289 creping agents could be treated prior toand/or subsequent to reduction or removal of CPD-forming species with abase ion exchange column, such as disclosed in U.S. Pat. No. 5,516,885and WO 92/22601; with carbon adsorption, such as disclosed in WO93/21384; membrane separation, e.g., ultrafiltration; extraction, e.g,ethyl acetate, such as disclosed in U.S. Statutory InventionRegistration H1613; or biodehalogenation, such as disclosed in U.S. Pat.No. 5,972,691, WO 96/40967 and U.S. Pat. Nos. 5,470,742, 5,843,763 and5,871,616, as well as U.S. application Ser. No. 09/629,629. Thedisclosures of each of these documents is incorporated by reference intheir entireties. Moreover, any combination of CPD-forming speciesreduction or removal as disclosed in the above-noted U.S. patentapplication Ser. Nos. 09/592,681, 09/363,224, and 09/330,200, each ofwhich is incorporated by reference in its entirety, can be utilized withthe enzymatic treatment for reduction and/or removal of CPD-formingspecies.

[0115] Still further, in accordance with the present invention, it isfurther noted that the enzymatic treatment to remove or reduceCPD-forming species can be performed in an overlapping manner withbiodehalogenation, or can be performed simultaneously with thebiodehalogenation. Thus, the present invention also relates to acombined process in which both enzymatic release of 3-CPD from resins isstarted, and simultaneously reduction of nitrogen-free organohalogencompounds occurs.

[0116] It is further noted that, in addition to the enzymatic treatmentfollowed by the biodehalogenation treatment, the two treatments can bedone simultaneously (aka combined treatment). “Simultaneously” meaningthe second treatment (either biodehalogenation or enzymatic) can bestarted before the first treatment (either biodehalogenation orenzymatic) is completed. For the present invention, the desiredviscosity is obtained by balancing the conditions of time, temperature,pH, enzyme concentration, starting viscosity, and solids content. Forexample, the combined treatment can be started at pH 6.8-7.8 for thefirst 4-24 hours and than lowered to pH of 5.5-7.0 or the pH can beallowed to drift down to 6.5-7.2 for the latter 8-48 hours of thecombined treatment. Preferred combined treatment conditions include, butare not limited to, pH 6.5 to 8.0, more preferably pH 6.8 to 7.6;preferred temperature range of 20° C. to 35° C., more preferably 25° C.to 33° C. Enzyme concentrations for combined treatment conditions willdepend upon its activity. For example, in the case of ALCALASE, theenzyme can be present in an amount of about 1 g of ALCALASE 2.5L type DX(as received) to 1600 g polyamine-epichlorohydrin resin (dry basis) to 1g of ALCALASE 2.5L type DX (as received) to 1.5 gpolyamine-epichlorohydrin resin (dry basis), also the enzyme can bepresent in an amount of about 1 g of ALCALASE 2.5L type DX (as received)to 160 g polyamine-epichlorohydrin resin (dry basis) to 1 g of ALCALASE2.5L type DX (as received) to 4 g polyamine-epichlorohydrin resin (drybasis). It is preferred that the combined treatment, is completed in 48hours or less. It is more preferred that the combined treatment iscompleted in 24 hours or less. For creping aids, the solids level whenusing the combined treatment conditions can be lower than 15 weightpercent, typically 4-14.5 weight percent % and preferably from about 8wt % to about 14.5 wt %. The combined treatment for creping aids canalso be done at solid level of 15 wt % and above, the preferred totalsolids levels are 15 to 40 weight percent, preferably 18-35 weightpercent and even more preferably 18-28 weight percent. An additionalrange that can be used in the present invention is 15-30 weight percent.

[0117] The preferred total solids level when using the combinedtreatment conditions for wet strength resins with 15 wt percent orhigher is 15-40 weight percent, preferably 16-35 weight percent and evenmore preferably 18-28 weight percent. When doing the combined treatmentof wet strength resins with less than 15 wt percent the preferred rangesare from about 4 wt % to about 14.5 weight % and from about 8 wt % toabout 14.5 wt %.

[0118] Still further, the present invention enables biodehalogenation athigh total solids content, as well as combined enzymatic treatment toremove or reduce CPD-forming species and biodehalogenation at high totalsolids content, with the possibility to reduce process cycle time, andat the same time creating an optimized reactor volume usage when runningthe process in batch or (repeated) fedbatch mode.

[0119] Biodehalogenation can be achieved in various manners, such asdisclosed in any one of U.S. Pat. Nos. 5,470,742; 5,843,763 and5,871,616, and U.S. application Ser. No. 09/629,629, or previous basetreatment and biodehalogenation as disclosed in U.S. Pat. No. 5,972,691,and WO 96/40967, with or without a previous inorganic base treatment,wherein the resin composition may be reacted with a microorganism orenzyme in adequate quantities to process epihalohydrin hydrolyzates tovery low levels. Microorganisms use dehalogenase enzymes to liberatehalide ion from the epihalohydrin and haloalcohol and then use furtherenzymes to break down the reaction products ultimately to carbon dioxideand water.

[0120] While not wishing to be bound by theory, it is noted that whenthe CPD-forming species is removed or reduced, CPD is released from theoligomeric and/or polymeric component of the resin, and therefore CPD isa component of the resin solution. With this in mind, the resin ispreferably subjected to treatment to remove or reduce the CPD-formingspecies, and then the resin is biodehalogenated. In this manner,epihalohydrin and epihalohydrin hydrolyzate (also referred to ashydrolysis by-products), including released CPD, can be removed, such asby the biodehalogenation. However, the resin can be initially treated,such as by biodehalogenation, and then subjected to treatment to remove,inhibit and/or reduce the CPD-forming species. In particular, any CPDthat will be released by the treatment should be readily soluble, andcan therefore be at least partially washed away from the resin. Forexample, when the resin with released CPD is included in a paperproduct, the CPD can be at least partially washed out of the paperproduct, and, due to the treatment, the resin in the paper product willnot produce CPD or will not produce undesirable amounts of CPD.Moreover, as discussed above, the enzymatic treatment to remove orreduce CPD-forming species can be performed in anoverlapping/simultaneous manner with the biodehalogenation.

[0121] The biocatalyst may be provided in the form of either livingcells or as an immobilized, unrefined cell-free extract or refineddehalogenase. The term “biodehalogenation” refers to the dehalogenationof a nitrogen-free organohalogen compound using a biocatalyst.

[0122] As the biocatalyst capable of biodehalogenation, there can beutilized any microorganism that is capable of dehalogenatingnitrogen-free organohalogen compounds, preferably CPD and DCP, whileleaving nitrogen-containing cationic polymers substantially intactduring the dehalogenation of the nitrogen-free organohalogen compounds.Preferably, the microorganisms utilized are Agrobacterium radiobacter(HK7) or Arthrobacter histidinolovorans (HK1), and preferably there isutilized a two-component mixture of Agrobacterium radiobacter (HK7) andArthrobacter higtidinolovorans (HK1). When only CPD is present, it ispreferred to use a single microorganism, HK1. When only DCP is present,it is preferred to use a single microorganism, HK7. Although the preciseidentity of the enzymes which make the method operable has not beenmade, it is believed that the enzymes which effectuate the method belongto the class of enzymes termed “hydrogen halide lyase typedehalogenase”.

[0123] In particular, a number of bacterial strains are disclosed inU.S. Pat. Nos. 5,470,742, 5,843,763, 5,871,616, and 5,972,691, and U.S.application Ser. No. 09/629,629, the disclosures of which areincorporated by reference herein in their entireties. These bacterialstrains include microorganisms which contain dehalogenating enzymescapable of dehalogenating haloalcohols and epihalohydrins depositedunder NCIMB Deposit Accession Nos. 40271, 40272, 40273, 40274, 40313 and40383. NCIMB stands for “National Collection of Industrial and MarineBacteria”. NCIMB, located at 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland, UK is an organization in the United Kingdom responsible fordocumenting and retaining samples of bacteria submitted for patentapplication purposes. In patent matters, NCIMB will supply to interestedparties who so request, authentic samples of bacteria claimed in patentliterature. NCIMB 40271 (Arthrobacter species), 40272 (Agrobacteriumradiobacter HK7), 40273 (Burkholderia cepacia formerly known asPseudomonas cepacia), and 40274 (Arthrobacter histidinolovorans HK1)were deposited on Apr. 2, 1990. NCIMB 40383 (Rhodococcus species) wasdeposited on Mar. 11, 1991, and NCIMB 40313 (Burkholderia cepaciaformerly known as Pseudomonas cepacia), was deposited on Aug. 30, 1990.Thus, the microorganisms have been filed in a depository under theprovisions of the Budapest Treaty, and the strains will be irrevocablyand without restriction or condition released to the public upon theissuance of a patent.

[0124] Still further, it is noted that two bacterial strains, which wereisolated from soil samples and the consortium designated HKC, arepreferably used, i.e., Arthrobacter histidinolovorans (HK1) which wasdeposited with the Centraalbureau voor Schimmelcultures at Oosterstraat1, P.O. Box 273, 3740 AG BAARN, The Netherlands, as Accession Number CBS108919 on Jul. 10, 2000, and Agrobacterium radiobacter (HK7) which wasdeposited with the Centraalbureau voor Schimmelcultures at Oosterstraat1, P.O. Box 273, 3740 AG BAARN, The Netherlands, as Accession Number CBS108920 on Jul. 10, 2000. In patent matters, Centraalbureau voorSchimmelcultures, which is a depository in conformance with the BudapestTreaty, will supply to interested parties, who so request, authenticsamples of bacteria claimed in patent literature. Thus, themicroorganisms have been filed in a depository under the provisions ofthe Budapest Treaty, and the strains will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. It is noted that NCIMB 40272 and CBS 108920 are believed to beidentical microorganisms, and that NCIMB 40274 and CBS 108919 arebelieved to be identical microorganisms.

[0125] Each of these microorganisms is capable of degrading both 1,3-DCPand 3-CPD. Moreover, the Agrobacterium radiobacter (HK7) is able toreduce 1,3-DCP levels faster than Arthrobacter histidinolovorans HK1,while Arthrobacter histidinolovorans (HK1) showed a preference for 3-CPDdegradation. Thus, as disclosed in U.S. application Ser. No. 09/629,629,it is deemed that the best biodehalogenation performance is obtainedwhen both bacteria were present. To ensure that both bacteria arepresent in the biodehalogenation process, it is preferred to start theprocess with Agrobacterium radiobacter (HK7) and to subsequently add theArthrobacter histidinolovorans (HK1). This would especially be thesituation for starting up a continuous biodehalogenation process.

[0126] The microorganisms containing suitable enzymes are used todehalogenate the epihalohydrin hydrolyzates contained in the resincomposition with or without an initial inorganic base treatment. Theenzymes and microorganisms are maintained in a suitable concentration tosubstantially metabolize the hydrolyzates to chloride ion and ultimatelycarbon dioxide and water. Thus the concentration of hydrolyzates in theresin composition of the present invention after biodehalogenationtreatment is preferably less than about 100 ppm (parts per million byweight relative to the total weight of aqueous solution containingresins after the bioreaction step), more preferably less than about 50ppm (parts per million by weight relative to the total weight of aqueoussolution containing resins after the bioreaction step), more preferablyless than about 10 ppm (parts per million by weight relative to thetotal weight of aqueous solution containing resins after the bioreactionstep), more preferably less than about 5 ppm (parts per million byweight relative to the total weight of aqueous solution containingresins after the bioreaction step), and even more preferably less thanabout 1 ppm (parts per million by weight relative to the total weight ofaqueous solution containing resins after the bioreaction step).

[0127] To achieve this, the concentration of microorganisms should be atleast about 5×10⁷ cells/ml, preferably at least about 10⁸ cells/ml andmost preferably at least about 10⁹ cells/ml. To maintain optimum activecontent of cells in the reactor, the reaction is best carried out atabout 30° C.+/−5° C. in the presence of oxygen (eg., from about 5 toabout 100% DOT) and nutrients in a stirred tank reactor. As used herein,the term “DOT” refers to “dissolved oxygen tension” and is the amount ofoxygen, expressed as a percentage, dissolved in a given volume of waterrelative to oxygen-saturated water at the same temperature and pressure.In a continuous process, the residence time is controlled by flow rateand monitored to ensure complete reaction. Thus, at steady state theconcentration of epihalohydrin hydrolyzates in the reactor will be fromabout 1 to about 1000 ppm. In a batch or fedbatch mode, which can bepreferably repeated, complete reaction can be ensured by monitoring, forexample by GC analysis, to achieve the desired reduced level ofepihalohydrin hydrolyzates.

[0128] The method of biodehalogenation in accordance with the presentinvention is carried out by contacting a microorganism or cell-freeenzyme-containing extract with the aqueous composition containing theunwanted organohalogen contaminants. Such contact is typically achievedby forming a slurry or suspension of the microorganism or cell-freeextract in the aqueous composition, with sufficient stirring.

[0129] If desired, the microorganism or enzymes can be removed from theproduct stream by filtration, sedimentation, centrifugation or othermeans known to those skilled in the art. Alternatively themicroorganisms or enzymes can remain in the final product and optionallydeactivated by thermal sterilization (e.g., by treatment at 140° C. for20 seconds) or by the addition of a suitable concentration of a suitablebiocidal agent. Suitable biocidal agents can be readily selected bythose of ordinary skill in the art. Thus, deactivation of themicroorganism can be performed by reducing the pH of the aqueous mixtureto 2.8, then adding a proprietary biocidal agent (e.g. Proxell® BDbiocidal agent, which comprises 1,2-benzisothiazolin-3-one) insufficient quantity, normally 0.02% to 0.1%, based on the weight of theaqueous composition. The biocidal agent may be added along withpotassium sorbate.

[0130] The removal of the microorganism may be performed by one or moreof the steps of filtration, centrifugation, sedimentation, or any otherknown techniques for removing microbes from a mixture. Themicroorganisms mineralize the nitrogen-free organohalogen compounds,producing CO₂, water, and biomass, with no glycerol left in the resin.Where the biocatalyst is an immobilized dehalogenase, the product of thereaction is glycidol, which can be hydrolyzed to glycerol with animmobilized hydrolase.

[0131] A problem associated with the removal of the microbes from themixture is that intensive methods of separation, such asmicrofiltration, remove not only microbes but also particles of cationicpolymer, with the result that the wet strength properties are reduced,which is undesirable. Therefore, it is preferable to leave thedeactivated microorganism in the mixture to avoid the problem ofreducing wet strength properties.

[0132] It has unexpectedly been determined that resin compositionshaving high concentrations of solids, i.e., greater than 15 wt %, morepreferably greater than 20 wt %, preferably greater than 25 wt %, can bebiodehalogenated using microorganisms and/or enzymes, when the resincomprises tertiary amine-based resins, such as Kymene® 450, Crepetrol®A3025 or Crepetrol® 80E. In the past, secondary amine-based resins, suchas Kymene®557H, Kymene® 557LX, Kymene® SLX, Kymene® Plus are notefficiently biodehalogenated at concentrations of solids of 15 orgreater weight %. In the present invention, secondary amine-based resinscan be efficiently biodehalogenated at 15 or greater wt %. In addition,it has been found that Daniels resins can be biodehalogenated at 15 orgreater wt %.

[0133] With regard to Daniel's resins, it is noted that cationicwater-soluble resins, derived from the reaction of epihalohydrins, suchas epichlorohydrin, and polyalkylene polyamines, such as ethylenediamine(EDA), bis-hexamethylenetriamine (BHMT) and hexamethylenediamine (HMDA)have long been known. These polyalkylene polyamine-epihalohydrin resinsare described in patents such as U.S. Pat. No. 3,655,506 to J. M.Baggett, et al. and others such as U.S. Pat. Nos. 3,248,353 and2,595,935 to Daniel et al. from which their generic description as“Daniel's Resins” arises. The disclosures of these patents areincorporated by reference herein in their entireties.

[0134] The polyalkylene polyamine employed in the present invention canpreferably be selected from the group consisting of polyalkylenepolyamines of the formula:

H₂N—[CHZ—(CH₂)_(n)—NR—]_(x)—H

[0135] where: n=1-7, x=1-6, R=H or CH₂Y, Z═H or CH₃, and Y═CH₂Z, H, NH₂,or CH₃,

[0136] polyalkylene polyamines of the formula:

H₂N—[CH₂—(CHZ)_(m)—(CH₂)_(n)—NR—]_(x)—H

[0137] where: m=1-6, n=1-6, and m+n=2-7, R═H or CH₂Y, Z═H or CH₃, andY═CH₂Z, H, NH_(2,) or CH₃,and mixtures thereof.

[0138] Polyalkylene polyamine-epihalohydrin resins comprise thewater-soluble polymeric reaction product of epihalohydrin andpolyalkylene polyamine. In making Daniel's Resins the polyalkylenepolyamine is added to an aqueous mixture of the epihalohydrin so thatduring the addition the temperature of the mixture does not exceed 60°C. Lower temperatures lead to further improvements, though too low atemperature may build dangerously latent reactivity into the system. Thepreferred temperatures fall within the range of about 25° C. to about60° C. More preferred is a range of from about 30° C. to about 45° C.

[0139] Alkylation of the polyamine occurs rapidly proceeding to formsecondary and tertiary amines depending on the relative amounts ofepihalohydrin and polyamine. The levels of epihalohydrin and polyamineare such that between about 50% and 100% of the available amine nitrogensites are alkylated to tertiary amines. Preferred levels are betweenabout 50% and about 80% alkylation of the amine nitrogen sites. Excessepihalohydrin beyond that required to fully alkylate all the amine sitesto the tertiary amine is less preferred because this results inincreased production of epihalohydrin byproducts.

[0140] Following complete addition of the polyamine, the temperature ofthe mixture is allowed to rise and /or the mixture is heated to effectcrosslinking and azetidinium formation. The crosslinking rate is afunction of concentration, temperature, agitation, and the additionconditions of the polyamine, all of which can be readily determined bythose skilled in the art. The crosslinking rate can be accelerated bythe addition of small shots of the polyamine or other polyamines of thepresent invention or addition of various alkalies at or near thecrosslinking temperature.

[0141] The resin can be stabilized against further crosslinking togelation by addition of acid, dilution by water, or a combination ofboth. Acidification to pH 5.0 or less is generally adequate.

[0142] The preferred polyamines are bishexamethylenetriamine,hexamethylenediamine, and their mixtures.

[0143] While not wishing to be bound by theory, it is noted that resinssuch as Kymene® at high solids concentrations have difficulty and areless easily biodehalogenated at high solids concentrations, such asabove 15 percent total solids due to viscosity increase and gelling ofthe resin resulting in reduced growth for the bacteria and loss ofproduct functionality due to crosslinking (=loss of functional groups).By controlling conditions, including pH, time, temperature,concentration of microorganism or enzyme, biodehalogenation can beachieved for Daniels resins and tertiary amine-based resins at, highertotal solids can be achieved. Preferred conditions for Daniels resins,for example Kymene® 736, are total solids level of 15 to 40 weightpercent, preferably 18-30 weight percent, even more preferably 18-22weight percent. Preferred conditions for tertiary amine-base resins aretotal solids level of 15 to 40 weight percent, preferably 18-35 weightpercent and even more preferably 18-28 weight percent. Preferred pHranges for both Daniels resins and tertiary amine-base resins are pH of5.0 to 8.0, more preferably pH ranges of 5.5 to 7.5. Preferredtemperature ranges for both Daniels resins and tertiary amine-baseresins are 20 to 40 degrees C., more preferably 25 to 35 degrees C. Itis preferred that the biodehalogenation step, starting for inoculation,is completed in 48 hours or less. It is more preferred that thebiodehalogenation step is completed in 24 hours of less from startingfor the inoculation.

[0144] It was not expected that biodehalogenation could be accomplishedat high solids concentration due to lack of water for themicroorganisms, higher osmotic pressure for higher solids content, andundefined problems, such as concentration of low molecular weightspecies. Moreover, it would be expected that pretreatment to removehigher residuals may be needed, such as by dilution or filtration.Moreover, it would not be expected that biodehalogenation could beachieved in a reasonable period of time, such as within 48 hours. Stillfurther, there would be an expectation of storage instability at highsolids concentrations; however, the resin compositions according to thepresent invention are storage stable, and are not susceptible togelling. The advantages of the present invention are obtained for highsolids whether or not the resin composition is treated to remove orreduce CPD-forming species.

[0145] Still further, in present invention, high-solids, wet-strengthresins can be biodehalogenated. Additionally, the enzymatic treatmentcan be done simultaneously with the biodehalogenation treatment.Although resins based on prepolymers without endcapping, such as KymeneE7219, can be biodehalogenated or enzymatically treated duringbiodehalogenation, it is preferred that these wet-strength resins areend-capped resins as described in WO 99/09252 and U.S. Pat. No.6,222,006, which are incorporated herein by reference in entirety. Whilenot wishing to be bound by theory, it is noted that the end-capper ispreferably is not an inhibitor of biodehalogenation. For example,residual hexanoic acid from the production of an end-capped prepolymerinhibits the microbial biodehalogenation, while residual acetic aciddoes not inhibit the microbial biodehalogenation. The preferred solidslevel for wet strength resins is 15 to 40 weight percent, preferably16-35 weight percent and even more preferably 18-28 weight percent. Anadditional range that can be used in the present invention is 15-30weight.

[0146] In order to more clearly describe the present invention, thefollowing non-limiting examples are provides for the purpose ofrepresentation, and are not to be construed as limiting the scope of theinvention. All parts and percentages in the examples are by weightunless indicated otherwise. Moreover, ND in the Examples indicates “NotDetected”.

EXAMPLES

[0147] Unless otherwise noted, Brookfield Viscosity was determined witha Brookfield LVDV-II+ Programmable Viscometer at 25° C. The procedureused was based on the Operating Instructions, Manual No. M/97-164. ThisViscometer will determine viscosity only if the correct spindle and rpmis used for the viscosity of the sample. Unless otherwise noted all CPDand DCP measurements are on a wet basis.

Example 1 Synthesis of a High-solids Polyaminopolyamide Resin

[0148] A 3-L round-bottom flask was fitted with a condenser, a pH meter,a temperature controlled circulating bath, an addition funnel and amechanical stirrer. To the kettle was added 775.0 g of 49.6 wt % aqueouspoly(adipic acid-co-diethylenetriamine) (available from HerculesIncorporated) and 505.3 g of water. The solution was heated to 25° C.and then 162.5 g of epichlorohydrin (Aldrich, 99%) was added over about2 minutes. The temperature was allowed to increase to 40° C. and wasmaintained at this temperature. 2.45 hours after the addition of theepichlorohydrin, 1046.5 g of water was added and the reaction mixturewas heated. After the reaction mixture reached 50° C. (20 minutes), 7.54g of 96% sulfuric acid was added. The temperature was raised to 70° C.and the Gardner-Holdt viscosity at 25° C. was monitored. After theGardner-Holdt viscosity reached G, the reaction was quenched by theaddition 187.5 g of water containing 12.90 g of 96% sulfuric acid. Thereaction mixture was allowed to cool to 25° C. The pH was adjusted to3.5 with an additional 3.00 grams of 96% sulfuric acid. The resin had21.08% total solids and a Brookfield viscosity of 153 cps.

Example 2 Enzyme-treatment of a Polyaminopolyamide-epi Resin (Example 1)

[0149] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 200.0 g of Example 1. The pH was raisedto 7.58 with 4.88 g of 30% aqueous sodium hydroxide. A 5 g aliquot wasremoved, the pH lowered to about 3 with 96% sulfuric acid and analyzedby GC. Then 5.18 g of ALCALASE 2.5 L type DX (available from Novozymes,used as received) was added. The temperature was raised from 21° C. to30° C. within 15 minutes and the Gardner-Holdt viscosity at 25° C. wasmonitored. Five gram aliquots of the reaction mixture were removed andthe pH lowered to about 3 with 96% sulfuric acid at 1, 2, 4, 6 and 8hours after the addition of ALCALASE and analyzed by GC. The pH wasadjusted to 7.5 at 2 hours with 0.27 g of 30% aqueous sodium hydroxideand at 4 hours with 0.18 g of 30% aqueous sodium hydroxide. After 8hours, the pH was lowered to 3.4 by addition of 2.22 g of 96% sulfuricacid. The resin had a Brookfield viscosity of 95 cps (at 25° C.).

Example 3 Biodehalogenation of Example 2

[0150] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 100.0 g of Example 2 and55.56 g of water. The pH was raised to 5.9 with 2.24 g of 30% aqueoussodium hydroxide and then 7.28 g of a blend of microorganisms comprisingan inoculum from a biodehalogenated polyaminopolyamide-epichlorohydrinresin. This represents a starting value of cell concentration of fromabout 10⁵ to about 10⁶ cells/ml. This starting value corresponds to afinal treatment level of about 10⁹ cells/ml as the process proceeds. Theinoculum was added, together with 1.36 g of a nutrient solution. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The microorganisms used were:Arthrobacter histidinolovorans (HK1) and Agrobacterium radiobacter(HK7). The air sparge was started, the temperature was maintained at 30°C. and the pH was maintained at 5.8 by periodic addition of 30% aqueoussodium hydroxide. After 48 hours, the mixture was cooled to roomtemperature and the pH was adjusted to 3.0 with 0.97 g of 96% sulfuricacid and 2.05 g of biocide solution was added. [The biocide solutionconsisted of 10% active Proxel® BD (from Zeneca Biocides) and 1.67%potassium sorbate in deionized water.] The resin had a total solids of14.5 wt. % and had a Brookfield viscosity of 62 cps (at 25° C.).

[0151] Acid Test

[0152] The amount of CPD producing species of this was estimated usingthe following acid test. A portion of resin to be tested was chargedinto a bottle containing a magnetic stirrer. The pH was adjusted to 1.0with 96% sulfuric acid. The bottle was capped and placed in a 50° C.water bath and maintained at 50° C. with stirring. Periodically,aliquots were removed from the bottle and submitted for GC analysis. TheCPD produced after 24 hours is used to estimate the amount of CPDproducing species. See Table 1 for results. TABLE 1 Gardner- Resin TempTime Holdt Epi 1,3-DCP 2,3-DCP 3-CPD Information (° C.) (hours)Viscosity (ppm) (ppm) (ppm) (ppm) Example 2a 21 0 N 15 1746 1.3 276Example 2b 30 1 F-G 20 2004 1.6 478 Example 2c 30 2 E-F 22 1720 1.7 508Example 2d 30 4 D-E 24 1802 1.4 680 Example 2e 30 6 D-E 26 1753 1.5 664Example 2f 30 8 E-F — — — — Example 3 — — — 0.1 ND 0.8 ND Acid Test 5024 — ND ND 0.10 0.66

Comp. Example 4 Enzyme-treatment of a Polyaminopolyamide-epi Resin

[0153] Enzyme-treatment: A portion of Example 1 was diluted to 13.5%total solids. A 1-L round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 900.0 g of the 13.5% Example 1. The pHwas raised to 7.54 with 13.85 g of 30% aqueous sodium hydroxide. A 5 galiquot was removed, the pH lowered to about 3 with 96% sulfuric acidand analyzed by GC. Then 15.0 g of AlCALASE 2.5 L type DX (availablefrom Novozymes, used as received) was added. The temperature was raisedfrom 22° C. to 35° C. within 15 minutes and the Gardner-Holdt viscosityat 25° C. was monitored. Five gram aliquots of the reaction mixture wereremoved and the pH lowered to about 3 with 96% sulfuric acid at 1, 2, 4,6 and 8 hours after the addition of ALCALASE and analyzed by GC. The pHwas adjusted to 7.5 at 2 hours with 0.80 g of 30% aqueous sodiumhydroxide, at 4 hours with 0.48 g of 30% aqueous sodium hydroxide and at6 hours with 0.78 g of 30% aqueous sodium hydroxide. At 6 hours, thetemperature was increased to 38° C. After 8 hours, the pH was lowered to3.5 by addition of 6.54 g of 96% sulfuric acid. The resin had aBrookfield viscosity of 32 cps (at 25° C.).

[0154] Biodehalogenation: A 1-L round-bottom flask was fitted with acondenser, a pH meter, a temperature controlled circulating bath, an airsparge tube and a mechanical stirrer. To the flask was added 700.0 g ofthe resin produced above. The pH was raised to 5.9 with 8.21 g of 30%aqueous sodium hydroxide and then 77.8 g of a blend of microorganismscomprising an inoculum from a biodehalogenatedpolyaminopolyamide-epichlorohydrin resin. This represents a startingvalue of cell concentration of from about 10⁵ to about 10⁶ cells/ml.This starting value corresponds to a final treatment level of about 10⁹cells/ml as the process proceeds. The inoculum was added, together with6.12 g of a nutrient solution. (The nutrient solution consisted of 8026ppm of potassium dihydrogen phosphate, 27480 ppm of urea, 4160 ppm ofmagnesium sulfate and 840 ppm of calcium chloride in tap water.) Themicroorganisms used were: Arthrobacter histidinolovorans (HK1) andAgrobacterium radiobacter (HK7). The air sparge was started, thetemperature was maintained at 30° C. and the pH was maintained at 5.8 byperiodic addition of 30% aqueous sodium hydroxide. After 48 hours, themixture was cooled to room temperature and the pH was adjusted to 3.0with 4.02 g of 96% sulfuric acid and 8.42 g of biocide solution wasadded. [The biocide solution consisted of 10% active Proxel® BD (fromZeneca Biocides) and 1.67% potassium sorbate in deionized water.] Theresin had a total solids of 14.77 wt. % and had a Brookfield viscosityof 61 cps (at 25° C.).

Example 5 Handsheet Evaluation of Example 3 and Comp. Example 4

[0155] Paper handsheets were prepared on a Noble and Wood handsheetmachine at pH 7.5 with 50:50 Rayonier bleached Kraft:Crown Vantagebleached hardwood Kraft dry lap pulp refined to 500 mL Canadian standardfreeness. Sheets were generated having 40 lb/3000 sq. ft. basis weightcontaining 0.5-1.0% of treated resin (based on the solids of untreatedresin). Handsheets were wet pressed to 33% solids and dried on a drumdrier at 230° C. for 55 seconds to give 3-5% moisture. The paper wasconditioned according to TAPPI Method T-402 and tested. Dry tensilestrength was determined using TAPPI Method T-494. Wet tensile strengthwas determined using TAPPI Method T-456 with a two hour soak time. TheCPD in paper products was determined by the following procedure:

[0156] CPD in Paper Products Procedure

[0157] COLD Water Extraction of Paper

[0158] The sample is cut and extracted with water at 23° C. (±2° C.) for24 hours, mixing occasionally. After the extraction period, the extractis filtered if necessary.

[0159] Note: Make sure all the paper is immersed in the water.

[0160] Procedure

[0161] 1. Wearing protective gloves, cut the sample into small pieces(approximately 1 cm×1 cm), and collect in a plastic bag. Mix the pieceswell.

[0162] 2. Weigh 10 grams of sample, to the nearest 0.0001 g, and placein a conical flask.

[0163] 3. Add 200 mL reagent grade water and stopper the flask.

[0164] 4. Place the flask in a water bath for 24 hours at 23° C. (±2°C.).

[0165] 5. Decant the solution into a 250 mL volumetric flask. Ifnecessary, filter the preparation using a fritted glass filter funnelwith filter flask. Rinse the pieces twice with additional reagent gradewater and fill to the mark.

[0166] Apparatus

[0167] 1. Conical flask, wide neck with ground glass stopper

[0168] 2. Volumetric flask, 250 mL

[0169]3. Fritted glass filter funnel (available from Lab Glass cat#lG-7080-170), with filter flask, 500 mL

[0170] 4. Water bath, to keep constant temperature of 23° C. (±2° C.).

[0171] 5. Paper cutter or scissors

[0172] 6. Analytical balance, capable of weighing to the nearest 0.0001g.

[0173] Reagents

[0174] 1. Water, available from Burdick & Jackson, cat. #365-4

[0175] ECD Method—Ether Elution & Derivitization

[0176] The separation of the analytes from the aqueous extract takesplace through a liquid-liquid extraction column. DCP and 3-CPD arederivitized with heptafluro-butrylimadazole (HFBI) and analyzed by gaschromatography using a μ-electron capture detector (μ-ECD).

[0177] Procedure

[0178] 1. Pipette 20 mL of the extracted water solution into a 35-mLvial.

[0179] 2. Add 2.34 g of NaCl to the vial. Cap and shake well until NaCldissolves.

[0180] 3. Pour the solution onto an extrelut column and allow to sit for15 minutes.

[0181] 4. After the waiting period, elute with 250 mL of eluant solution(95% diethyl ether/isooctane. (Collect the eluant in a volumetric flask)

[0182] 5. Pour the eluant into a 500 mL round bottom flask

[0183] 6. The solvent is removed using a rotary evaporator (Note: thevacuum is not to exceed 200 mm Hg), until about 15 mL remains.

[0184] 7. Pipette 1 mL Internal Standard solution into the remainingiso-octane.

[0185] 8. A method blank of reagent grade water prepared according tosteps 2 to 7 must also be run to check for interference

[0186] Derivitization

[0187] 1. Using a syringe or micropipette, add 200 μL of HFBI to theflask. Stopper the flask and swirl the solution to mix well.

[0188] 2. Let the flask stand for 15 minutes at room temperature.

[0189] 3. Quantitatively transfer the solution to a 25 mL mixingcylinder and fill to the mark with iso-octane.

[0190] 4. Add ˜1.5 mL reagent grade water to the volumetric, stopper andshake to mix well. A precipitate will have formed but will disappearwhen mixed well with the water.

[0191] 5. After the phase separation, remove approximately 20 mL of theorganic phase and put in a 30 mL glass vial, which contains 2 mL reagentgrade water. Shake vigorously for 1 minute.

[0192] 6. After phase separation. Remove the water layer and discard.The organic phase will be analyzed by gas chromatography using aμ-Electron Capture detector (ECD).

[0193] Reagents

[0194] 1. Di-ethyl ether, available from FLUKA, P.O. Box 355, Milwaukee,Wis. Cat. No. 31690.

[0195] must use a.p. quality; the ether may be neither dried norstabilized with ethanol.

[0196] 1. Water, available from Burdick & Jackson, cat. #365-4

[0197] 2. Sodium Chloride

[0198] 3. 1,3-DCP; available TCI Americas, Cat. No. D0402.

[0199]4. 3-CPD; available Aldrich, Cat. No. 10227-1.

[0200] 5. Acetonitrile, Nanograde; available Fisher, Cat. No. 2442.

[0201] 6. Iso-octane, EM Science, Cat. No. TX1389

[0202] 7. Eluant: 95 mL di-ethyl ether/5 mL iso-octane.

[0203] 8. Heptaflurobutyrylimadozole (HFBI), available from Pierce, Cat.No. 44211

[0204] 9. 3-methoxy-1,2-propanediol (internal standard)

[0205] 10. Solid phase extraction column, Supelco, Supelco Park,Bellefonte, Pa. 16823-0048, prepared according to Section XXX. Cat. No.57022.

[0206] 11. Varian Hydromatrix; available Varian, Inc., Cat. No.00198003.

[0207] Apparatus

[0208] 1. Gas Chromatograph, Hewlett Packard Model 5890, or equivalent,capable of linear column temperature programming, and equipped with aμ-Electron Capture detector (μ-ECD).

[0209] 2. Data handling system, Hewlett Packard ChemStation orequivalent.

[0210] 3. Chromatographic column, DB-5MS, 60 meters×0.25 mmI.D.—available from J & W Scientific Inc., 91 Blur Ravin Road, Folsom,Calif. 95630, Cat. No. 122-5562.

[0211] 4. Flasks, volumetric, glass stoppered, 5 mL, 10 mL, 25 mL, 50mL, 100 mL, 250 mL.

[0212] 5. Vials, glass with teflon-lined screw caps, 17 mL, 30 mL, 4 oz.

[0213] 6. Pipettes, transfer, 0.5, 1, 2, 3, 5, 10, 20 mL Class A.

[0214] 7. Medicine droppers, glass—Fisher, Cat. No. 13-701

[0215] 8. Analytical balance, capable of weighing to the nearest 0.0001g.

[0216] 9. Solid phase extraction column, Supelco, Supelco Park,Bellefonte, Pa. 16823-0048, prepared according to Section XXX. Cat. No.57022.

[0217] 10. Glass wool

[0218] 11. 500 mL round bottom flask with stopper, available from LabGlass, Cat. No. 013 and Cat. No. 114.

[0219] 12. Rotary Vacuum evaporator operating at 35-40° C./800 mbar

[0220] 13. 500 μL syringe or disposable micro-pipettes

[0221] 14. Type A mixing cylinders, 25 mL; available Fisher, Cat. No.08-563-1F.

[0222] Internal Standard Solution (Low Level)

[0223] 1. Weigh 50 mg 3-methoxy-1,2-propanediol into a 50-mL volumetricflask and record the weight to the nearest 0.0001 g.

[0224] 2. Dilute to the mark with acetonitrile.

[0225] 3. Pipette 0.25 mL of solution in step 2 into a 100-mL volumetricflask and dilute to volume with diethyl ether.

[0226] 4. Pipette 10.0 mL of solution in step 3 into a 100 mL volumetricand dilute to volume with diethyl ether.

[0227] 1,3-DCP, 3-CPD Calibration Solution (Low Level)

[0228] 1. Weigh 50 mg 1,3-dichloro-2-propanol into a 50-mL volumetricflask and record the weight to the nearest 0.0001 g.

[0229] 2. Dilute to the mark with acetonitrile.

[0230] 3. Pipette 0.5 mL of solution in step 2 into a 10-mL volumetricflask and dilute to volume with diethyl ether.

[0231] 4. Weigh 50 mg 3-chloro-1,2-propanediol into a 50-mL volumetricflask and record the weight to the nearest 0.0001 g.

[0232] 5. Dilute to the mark with acetonitrile.

[0233] 6. Pipette 0.5 mL of solution in step 5 into a 10-mL volumetricflask and dilute to volume with diethyl ether.

[0234] 7. Combine solutions in step 3 and step 6 in a 30-mL vial and mixwell.

[0235] 8. Pipette 2.5 mL of solution in step 7 into a 100-mL volumetricflask and dilute to volume with diethyl ether.

[0236] 9. Pipette 10.0 mL of solution in step 8 into a 100-mL volumetricflask and dilute to volume with diethyl ether. This is the CalibrationStock Solution.

[0237] Calibration Curves (Low Level):

[0238] 1. Pipette 0.1 mL of the Calibration Stock Solution into a 25-mLvolumetric flask containing 1.0 mL of the Internal Standard Solution.Using a pipette, add 5.9 mL of diethyl ether to the flask. This will becalibration Level #1.

[0239] 2. Pipette 0.2 mL of the Calibration Stock Solution into a 25-mLvolumetric flask containing 1.0 mL of the Internal Standard Solution.Using a pipette, add 5.8 mL of diethyl ether to the flask. This will becalibration Level #2.

[0240] 3. Pipette 0.5 mL of the Calibration Stock Solution into a 25-mLvolumetric flask containing 1.0 mL of the Internal Standard Solution.Using a pipette, add 5.5 mL of diethyl ether to the flask. This will becalibration Level #3.

[0241] 4. Pipette 1.0 mL of the Calibration Stock Solution into a 25-mLvolumetric flask containing 1.0 mL of the Internal Standard Solution.Using a pipette, add 5.0 mL of diethyl ether to the flask. This will becalibration Level #4.

[0242] 5. Pipette 2.0 mL of the Calibration Stock Solution into a 25-mLvolumetric flask containing 1.0 mL of the Internal Standard Solution.Using a pipette, add 4.0 mL of diethyl ether to the flask. This will becalibration Level #5.

[0243] 6. Add 15 mL iso octane to each of the volumetric flasks fromsteps 1 through 6.

[0244] 7. Using a syringe, add 200 μL HFBI to each of the volumetricflasks from step 7, then stopper and allow to stand at room temperaturefor 15 minutes with occasional shaking.

[0245] 8. Dilute each flask to a final volume of 25-mL with iso-octane.

[0246] 9. Add ˜1.5 mL reagent grade water to each volumetric, stopperand shake to mix well. A precipitate will have formed but will disappearwhen mixed well with the water.

[0247] 10. After the phase separation, transfer approximately 20 mL ofthe organic phase to a 30-mL glass vials in which each contain 2 mLreagent grade water. Shake vigorously for 1 minute.

[0248] 11. After phase separation. Remove the water layer and discard.The organic phases will be analyzed by gas chromatography using aμ-Electron Capture detector (μ-ECD) to determine the calibration curve.

[0249] GC Operating Conditions Column Temperatures Initial  50° C.Initial hold time 2 min Initial rate 1.5° C./min 2^(nd) temp 100° C.2^(nd) hold  5 min 2^(nd) rate  25° C./min Final 300° C. Final hold time10 min Inlet 250° C. Detector temp 320° C. Flow Rates Helium (carriergas) 1.5 mL/min at 20 psi (column head pressure at 35° C.) Argon/Methane60 mL/min

[0250] Section XXX

[0251] Preparing the Extrelut QE Columns

[0252] 1. Using a solid phase extraction reservoir, push approximately0.5 g of glass wool to the bottom.

[0253] 2. Weigh 18 g Varian Hydromatrix and pour into reservoir. Using aglass probe, pack extrelut tightly.

[0254] 3. Place approximately 0.5 g glass wool on top of reservoir.

[0255] Results are reported in Table 2. The data show that enzymetreatment at 21% solids gave essentially the same results as treatmentat 13.5% solids. These results allow for a more economical enzymatictreatment. TABLE 2 Natural Aged Paper Results Natural Aged Paper BasisWt. Normalized % Dry Wet % % of Resin Tensile Tensile wet/ Comp. CPD inExample Added (lbs/in) (lbs/in) dry Ex XX Paper (ppb) Blank — 17.55 0.533 — <3 Comp. Ex. 0.25 24.47 3.89 16 — <3 4a Comp. Ex. 0.50 24.61 4.86 20— <3 4b Comp. Ex. 1.00 24.65 5.70 23 — 10 4c Example 3a 0.25 22.58 3.6016 93 <3 Example 3b 0.50 23.88 4.63 19 95 <3 Example 3c 1.00 24.72 5.3822 94  7

Example 6-17 General Procedure for Enzyme-treatment of aPolyaminopolyamide-epi Resins

[0256] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 400.0 g of Kymene® E7219 (Available fromHercules Incorporated, Wilmington, Del.; 21.51% solids, 267 cpsBrookfield viscosity at 25° C.). The pH was increased with 30% aqueoussodium hydroxide. A 5 g aliquot was removed, the pH lowered to about 3with 96% sulfuric acid and analyzed by GC. ALCALASE 2.5 L type DX(available from Novozymes, used as received) was added (amount indicatedin Table 3). The temperature was raised within 15 minutes to the desiredtreatment temperature and the Gardner-Holdt viscosity at 25° C. wasmonitored. Five gram aliquots of the reaction mixture were removed andthe pH lowered to about 3 with 96% sulfuric acid at 1, 2, 4, 6 and 8hours after the addition of ALCALASE and analyzed by GC. The pH waschecked every hour and was adjusted with 30% aqueous sodium hydroxide ifthe pH drifted by more than 0. 10. After 8 hours, the pH was lowered to3.5 by addition of 96% sulfuric acid. If the Gardner-Holdt viscosityreading was in-between letters, both letters are recorded in the Table.If the viscosity was increasing more than desired, the pH adjustmentswith 30% aqueous sodium hydroxide were discontinued. If the viscosityincreased to the point of risking gelation, the pH was lowered to 3.5 byaddition of 96% sulfuric acid. BV (cps) is the Brookfield Viscosity(measured at 25° C.) of the final resin. The ALCALASE:Active solidsratio is defined as the amount of ALCALASE 2.5 L type DX, used asreceived, compared to the amount of active solids in the resin. SeeTable 3 for details.

Example 18-19 General Procedure for Enzyme-treatment of aPolyaminopolyamide-epi Resins

[0257] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 200.0 g of Example 1. The pH wasincreased with 30% aqueous sodium hydroxide. A 4 g aliquot was removed,the pH lowered to about 3 with 96% sulfuric acid and analyzed by GC.ALCALASE 2.5 L type DX (available from Novozymes, used as received) wasadded (amount indicated in Table 3). The temperature was raised within15 minutes to the desired treatment temperature and the Gardner-Holdtviscosity at 25° C. was monitored. Four gram aliquots of the reactionmixture were removed and the pH lowered to about 3 with 96% sulfuricacid at 1, 2, 4, 6 and 8 hours (if non-gelled) after the addition ofALCALASE and analyzed by GC. The pH was checked every hour and wasadjusted with 30% aqueous sodium hydroxide if the pH drifted by morethan 0.10. After 8 hours, the pH was lowered to 3.5 by addition of 96%sulfuric acid. If the Gardner-Holdt viscosity reading was in-betweenletters, both letters are recorded in Table 3. If the viscosity wasincreasing more than desired, the pH adjustments with 30% aqueous sodiumhydroxide were discontinued. With both reactions, the viscosity wasallowed to increase to the point of gelation. BV (cps) is the BrookfieldViscosity (measured at 25° C.) of the final resin. The ALCALASE:Activesolids ratio is defined as the weight amount of ALCALASE 2.5 L type DX,used as received, compared to the weight amount of active solids in theresin. See Table 3 for details. TABLE 3 Gardner- Resin ALCALASE: TempTime Holdt BV Example Active Solids pH (° C.) (hours) Viscosity (cps)Example 6a 1.0:8.3  7.2 21 0 I-J Example 6b 1.0:8.3  7.2 30 1 E-FExample 6c 1.0:8.3  7.2 30 2 D-E Example 6d 1.0:8.3  7.2 30 4 D Example6e 1.0:8.3  7.2 30 6 D Example 6f 1.0:8.3  7.2 30 8 D 84 Example 7a1.0:8.3  7.8 21 0 I-J Example 7b 1.0:8.3  7.8 30 1 E-F Example 7c1.0:8.3  7.8 30 2 E-F Example 7d 1.0:8.3  7.8 30 4 E-F Example 7e1.0:8.3  7.8 30 6 F-G Example 7f 1.0:8.3  7.8 30 8 H-I 255 Example 8a1.0:16.6 7.2 22 0 I-J Example 8b 1.0:16.6 7.2 25 1 G-H Example 8c1.0:16.6 7.2 25 2 G Example 8d 1.0:16.6 7.2 25 4 F Example 8e 1.0:16.67.2 25 6 E-F Example 8f 1.0:16.6 7.2 25 8 E-F 105 Example 9a 1.0:16.67.8 22 0 I-J Example 9b 1.0:16.6 7.8 25 1 G-H Example 9c 1.0:16.6 7.8 252 G-H Example 9d 1.0:16.6 7.8 25 4 I Example 9e 1.0:16.6 7.8 25 6 J-KExample 9f 1.0:16.6 7.8 25 7 L-M 379 Example 10a 1.0:11.1 7.2 23 0 I-JExample 10b 1.0:11.1 7.2 25 1 F-G Example 10c 1.0:11.1 7.2 25 2 FExample 10d 1.0:11.1 7.2 25 4 E Example 10e 1.0:11.1 7.2 25 6 D-EExample 10f 1.0:11.1 7.2 25 8 D 77 Example 11a 1.0:11.1 7.8 23 0 I-JExample 11b 1.0:11.1 7.8 25 1 F-G Example 11c 1.0:11.1 7.8 25 2 FExample 11d 1.0:11.1 7.8 25 4 E-F Example 11e 1.0:11.1 7.8 25 6 FExample 11f 1.0:11.1 7.8 25 8 F 149 Example 12a 1.0:8.3  7.2 22 0 H-IExample 12b 1.0:8.3  7.2 25 1 F-G Example 12c 1.0:8.3  7.2 25 2 E-FExample 12d 1.0:8.3  7.2 25 4 D-E Example 12e 1.0:8.3  7.2 25 6 CExample 12f 1.0:8.3  7.2 25 8 B-C 60 Example 13a 1.0:8.3  7.8 22 0 H-IExample 13b 1.0:8.3  7.8 25 1 F-G Example 13c 1.0:8.3  7.8 25 2 E-FExample 13d 1.0:8.3  7.8 25 4 D-E Example 13e 1.0:8.3  7.8 25 6 CExample 13f 1.0:8.3  7.8 25 8 C-D 85 Example 14a 1.0:8.3  7.2 22 0 H-IExample 14b 1.0:8.3  7.2 35 1 F-G Example 14c 1.0:8.3  7.2 35 2 B-FExample 14d 1.0:8.3  7.0 35 4 F Example 14e 1.0:8.3  6.9 35 6 F-CExample 14f 1.0:8.3  6.8 35 8 G-H 175 Example 15a 1.0:8.3  7.5 22 0 H-IExample 15b 1.0:8.3  7.5 35 1 F-C Example 15c 1.0:8.3  7.5 35 2 FExample 15d 1.0:8.3  7.3 35 4 J-K Example 15e 1.0:8.3  7.3 35 4.5 N 434Example 16a 1.0:16.6 7.5 22 0 H-I Example 16b 1.0:16.6 7.5 25 1 CExample 16c 1.0:16.6 7.5 25 2 G Example 16d 1.0:16.6 7.5 25 4 F-CExample 16e 1.0:16.6 7.5 25 6 B-F Example 16f 1.0:16.6 7.5 25 8 B-F 138Example 17a 1.0:11.1 7.5 22 0 H-I Example 17b 1.0:11.1 7.5 25 1 F-CExample 17c 1.0:11.1 7.5 25 2 B-F Example 17d 1.0:11.1 7.5 25 4 D-EExample 17e 1.0:11.1 7.5 25 6 D Example 17f 1.0:11.1 7.5 25 8 D 80Example 18a 1.0:16.3 7.5 22 0 H Example 18b 1.0:16.3 7.5 33 1 I Example18c 1.0:16.3 7.5 33 2 K Example 18d 1.0:16.3 7.5 33 3 V Example 18e1.0:16.3 7.5 33 4 W-X Example 19a 1.0:8.1  7.5 22 0 H Example 19b1.0:8.1  7.5 33 1 F Example 19c 1.0:8.1  7.5 33 2 B-F Example 19d1.0:8.1  7.5 33 4 F Example 19e 1.0:8.1  7.5 33 6 H Example 19f 1.0:8.1 7.5 33 7 L Example 19g 1.0:8.1  7.5 33 8 Gel

Example 20 Combined Enzyme-treatment and Biodehalogenation of aPolyaminopolyamide-epi Resin

[0258] Scale-up 1 (Starter Preparation)

[0259] A portion of Kymene® E7219 (Available from Hercules Incorporated,Wilmington, Del.; 21.51% solids, 267 cps Brookfield viscosity at 25° C.)was diluted to 13.5% total solids. A 400-mL round-bottom flask wasfitted with a condenser, a pH meter, a temperature controlledcirculating bath, an air sparge tube and a mechanical stirrer. To theflask was added 400 g of the 13.5% Kymene® E7219. The pH was raised to7.54 with 7.42 g of 30% aqueous sodium hydroxide. A 5 g aliquot wasremoved, the pH lowered to about 3 with 96% sulfuric acid and analyzedby GC. Then 3.33 g of AlCALASE 2.5 L type DX (available from Novozymes,used as received) was added and then 44.4 g of a blend of microorganismscomprising an inoculum from a biodehalogenatedpolyaminopolyamide-epichlorohydrin resin. This represents a startingvalue of cell concentration of from about 10⁵ to about 10⁶ cells/ml.This starting value corresponds to a final treatment level of about 10⁹cells/ml as the process proceeds. The inoculum was added, together with3.50 g of a nutrient solution. (The nutrient solution consisted of 8026ppm of potassium dihydrogen phosphate, 27480 ppm of urea, 4160 ppm ofmagnesium sulfate and 840 ppm of calcium chloride in tap water.) Themicroorganisms used were: Arthrobacter histidinolovorans (HK1) andAgrobacterium radiobacter (HK7). The air sparge was started, thetemperature was maintained at 30° C. The treatment was monitored byGardner-Holdt viscosity and the bacterial growth was monitored byoptical density (OD₆₀₀). OD₆₀₀ was determined by measuring the opticaldensity at a wavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. Periodically, 5g aliquot were removed, the pH lowered to about 3 with 96% sulfuric acidand analyzed by GC. The pH of the treatment was maintained for the first30 hours at 7.1-7.5 by periodic addition of 30% aqueous sodiumhydroxide. After 30 hours, the pH was lower to 5.8 by addition of 96%sulfuric acid. After 48 hours, the resulting mixture was used as theinoculum for Scale-up 2 below.

[0260] Scale-up 2

[0261] A portion of Kymene® E7219 (Available from Hercules Incorporated,Wilmington, Del.; 21.51% solids, 267 cps Brookfield viscosity at 25° C.)was diluted to 13.5% total solids. A 2-L round-bottom flask was fittedwith a condenser, a pH meter, a temperature controlled circulating bath,an air sparge tube and a mechanical stirrer. To the flask was added 1600g of the 13.5% Kymene® E7219. The pH was raised to 7.52 with 30.38 g of30% aqueous sodium hydroxide. A 5 g aliquot was removed, the pH loweredto about 3 with 96% sulfuric acid and analyzed by GC. Then 13.32 g ofAlCALASE 2.5 L type DX (available from Novozymes, used as received) wasadded and then 177.8 g of the inoculum from Scale-up 1 above was added,together with 14.0 g of a nutrient solution. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The treatment was monitored by Gardner-Holdt viscosity and thebacterial growth was monitored by optical density (OD₆₀₀). OD₆₀₀ wasdetermined by measuring the optical density at a wavelength of 600 nmusing a Spectronic® Genesys™ UV/Vis spectrophotometer (SpectronicInstruments, Incorporated, Rochester, N.Y., USA) and a disposable cuvetwith 1-cm pathlength. Periodically, 5 g aliquot were removed, the pHlowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH ofthe treatment was maintained for the first 8.5 hours at 7.2-7.5 byperiodic addition of 30% aqueous sodium hydroxide. For the remainingtreatment time, the pH was maintained at pH 6.8-7.2 by periodic additionof 30% aqueous sodium hydroxide. After 48 hours, the mixture was cooledto room temperature and the pH was adjusted to 2.8 with 12.80 g of 96%sulfuric acid and 19.26 g of biocide solution was added. [The biocidesolution consisted of 10% active Proxel® BD (from Zeneca Biocides) and1.67% potassium sorbate in deionized water.] See Table 4 for the resultsfrom monitoring the treatment. Table 4 pH Gardner OD₆₀₀ DCP CPD AliquotTime (30 C) Viscosity (abs.) (ppm) (ppm)  −1 “0” 7.49 (21 C) D/E 0.058679 204 −78A 0 Time “0” is right after NaOH addition, Time 0 is rightafter Alcalase addition. — 0.25 7.43 — — — —  −2 1 7.39 B/C — 589 242 −3 2 7.32 B/C 0.062 585 268  −4 4 7.32 B/C 0.067 586 309  −5 6 7.25 B0.064 584 322 — 7 7.20 to — — — — 7.52  −6 8 7.50 B 0.061 554 331  −7 107.40 B 0.064 540 363  −8 14 7.26 to B 0.064 519 399 7.45  −9 24 7.20 D0.109 476 384 — 28 7.09 E/F 0.165 −10 30 7.05 to G/H 0.201 422 296 5.79— 32 5.83 H 0.260 — — — 34 5.84 — 0.300 — — −11 37 5.81 H/I 0.328 360317 −12 48 5.60 I/J 0.473 ND 198 pH Gardner OD₆₀₀ DCP CPD Aliquot Time(30 C) Viscosity (abs.) (ppm) (ppm)  −1 “0” 7.48 (26 C) D/E 0.071 745256 −82A & 0 Time “0” is right after NaOH addition, −84B Time 0 is rightafter Alcalase addition. — 0.25 7.46 — —  −2 1 7.39 C/D — 639 394  −3 27.32 C/D 0.086 614 427  −4 4 7.20 to C 0.108 579 531 7.40  −5 6 7.31 C0.138 537 540  −6 8.5 7.16 B/C 0.198 391 629  −7 12.5 7.02 B/C 0.271 66796  −8 22 6.81 D 0.481 ND 144 — 24 6.78 to — — 7.12 Resin is lighttannish yellow. — 28 7.02 D/E 0.560  −9 30 6.99 D/E 0.578 0.3 7.8 −10 486.95 D/E 0.611 0.3 0.5 Acid Test — — — — ND 1.1

Example 21 Combined Enzyme-treatment and Biodehalogenation of aPolyaminopolyamide-epi Resin. (Using Twice the Alcalase as in Example20)

[0262] Scale-up 1 (Starter Preparation)

[0263] A portion of Kymene® E7219 (Available from Hercules Incorporated,Wilmington, Del.; 21.51% solids, 267 cps Brookfield viscosity at 25° C.)was diluted to 13.5% total solids. A 400-mL round-bottom flask wasfitted with a condenser, a pH meter, a temperature controlledcirculating bath, an air sparge tube and a mechanical stirrer. To theflask was added 400 g of the 13.5% Kymene® E7219. The pH was raised to7.52 with 7.38 g of 30% aqueous sodium hydroxide. A 5 g aliquot wasremoved, the pH lowered to about 3 with 96% sulfuric acid and analyzedby GC. Then 6.66 g of AlCALASE 2.5 L type DX (available from Novozymes,used as received) was added and then 44.4 g of a blend of microorganismscomprising an inoculum from a biodehalogenatedpolyaminopolyamide-epichlorohydrin resin. This represents a startingvalue of cell concentration of from about 10⁵ to about 10⁶ cells/ml.This starting value corresponds to a final treatment level of about 10⁹cells/ml as the process proceeds. The inoculum was added, together with3.50 g of a nutrient solution. (The nutrient solution consisted of 8026ppm of potassium dihydrogen phosphate, 27480 ppm of urea, 4160 ppm ofmagnesium sulfate and 840 ppm of calcium chloride in tap water.) Themicroorganisms used were: Arthrobacter histidinolovorans (HK1) andAgrobacterium radiobacter (HK7). The air sparge was started, thetemperature was maintained at 30° C. The treatment was monitored byGardner-Holdt viscosity and the bacterial growth was monitored byoptical density (OD₆₀₀). OD₆₀₀ was determined by measuring the opticaldensity at a wavelength of 600 nm using a Spectronic® Genesys™ TM UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. Periodically, 5g aliquot were removed, the pH lowered to about 3 with 96% sulfuric acidand analyzed by GC. The pH of the treatment was maintained for the first24 hours at 7.2-7.5 by periodic addition of 30% aqueous sodiumhydroxide. After 24 hours, the pH was allow to drift down to pH 6.71over the course of 24 hours. After 48 hours, the resulting mixture had aBrookfield viscosity of 71 cps (measured at 25° C.). This mixture wasused as the inoculum for Scale-up 2 below.

[0264] Scale-up 2

[0265] A portion of Kymene® E7219 (Available from Hercules Incorporated,Wilmington, Del.; 21.51% solids, 267 cps Brookfield viscosity at 25° C.)was diluted to 13.5% total solids. A 2-L round-bottom flask was fittedwith a condenser, a pH meter, a temperature controlled circulating bath,an air sparge tube and a mechanical stirrer. To the flask was added 1600g of the 13.5% Kymene® E7219. The pH was raised to 7.55 with 29.99 g of30% aqueous sodium hydroxide. A 5 g aliquot was removed, the pH loweredto about 3 with 96% sulfuric acid and analyzed by GC. Then 26.64 g ofAlCALASE 2.5 L type DX (available from Novozymes, used as received) wasadded and then 177.8 g of the inoculum from Scale-up 1 above was added,together with 14.0 g of a nutrient solution. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The treatment was monitored by Gardner-Holdt viscosity and thebacterial growth was monitored by optical density (OD₆₀₀). OD₆₀₀ wasdetermined by measuring the optical density at a wavelength of 600 nmusing a Spectronic® Genesys™ UV/Vis spectrophotometer (SpectronicInstruments, Incorporated, Rochester, N.Y., USA) and a disposable cuvetwith 1-cm pathlength. Periodically, 5 g aliquot were removed, the pHlowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH ofthe treatment was maintained for the first 8 hours at 7.1-7.5 byperiodic addition of 30% aqueous sodium hydroxide. For the remainingtreatment time, the pH was maintained at pH 6.8-7.2 by periodic additionof 30% aqueous sodium hydroxide. After 48 hours, the mixture was cooledto room temperature and the pH was adjusted to 2.8 with 12.85 g of 96%sulfuric acid and 19.26 g of biocide solution was added. [The biocidesolution consisted of 10% active Proxel® BD (from Zeneca Biocides) and1.67% potassium sorbate in deionized water.] The resin had a Brookfieldviscosity of 30 cps (measured at 25° C.). See Table 5 for the resultsfrom monitoring the treatment. TABLE 5 pH G/H OD₆₀₀ DCP CPD Sample Time(30 C) Viscosity (abs.) (ppm) (ppm)  −1 “0” 7.50 (21 C) D/E 0.060 770269 −80B 0 Time “0” is right after NaOH addition, Time 0 is right afterAlcalase addition. — 0.25 7.44 — — — —  −2 1 7.37 B — 657 412  −3 2 7.34B 0.058 645 477  −4 4 7.30 A/B 0.063 677 526  −5 6 7.25 A/B 0.062 623508 — 7 7.20 to — — — — 7.52  −6 8 7.50 A 0.059 599 504  −7 10 7.40A/A-1 0.062 615 559  −8 14 7.26 to A/A-1 0.065 592 569 7.47  −9 24 7.22A/B 0.139 516 560 — 28 7.12 A/B 0.259 — — −10 30 7.11 B 0.295 387 508 —32 7.07 B 0.336 — — — 34 7.03 — 0.391 — — −11 37 6.93 B 0.475 ND 497 −1248 6.71 C 0.716 ND ND Scale-up 2 pH G/H OD₆₀₀ DCP CPD Aliquot Time (30C) Viscosity (abs.) (ppm) (ppm)  −1 “0” 7.48 (26 C) D/E 0.101 778 237−84B 0 Time “0” is right after NaOH addition, Time 0 is right afterAlcalase addition. — 0.25 7.46 — —  −2 1 7.39 C — 641 384  −3 2 7.31 B/C0.129 583 433  −4 4 7.10 to B 0.199 507 531 7.40  −5 6 7.34 B 0.270 371562  −6 8.5 — A/B 0.422 96 653 Recalibration of the pH meter.  −7 12.56.75 to A 0.618 ND 303 7.14  −8 22 6.85 A/B 0.877 ND 0.5 — 24 6.85 to —— 7.09 Resin is dark orange, tannish-brown — 28 7.01 to A/B 0.932 7.10 −9 30 7.08 A/B 0.959 ND ND −10 48 6.84 B 1.080 ND ND Acid Test — — — —ND 0.1

[0266] Examples 20 and 21 clearly show that the enzyme treatment and thebiodehalogenation treatment can be effectively combined. When twice theAlcalase was used, a preferred balance of conditions allowed a preferredviscosity to be obtained.

Example 22 Alcalase-biodehalogenation of Kymene® E7219 (See Table 6 forData and Details)

[0267] Kymene® E7219 (Available from Hercules Incorporated, Wilmington,Del.; Zwijndrecht, Netherlands plant) was diluted to 13.40% and had aBrookfield viscosity of 76 cps. Pasteurization: A 3-L round-bottom flaskwas fitted with a condenser, a temperature controlled circulating bathand a mechanical stirrer. To the flask was added 2800 g of the resin.The pH was adjusted from 3.4 to 3.0 with concentrated sulfuric acid andwas heated over 15 min from 25° C. to 80° C. The resin was held at 80°C. for 15 minutes, cooled to 75° C. in 10 minutes and then cooled to 30°C. The pasteurized resin had a Brookfield viscosity of 48 cps and wasstored in sterile containers.

[0268] Sterilization of Kymene E7219

[0269] A 500 g portion of the Kymene E7219 was diluted to 8%, placed inan autoclavable bottle and heated in an autoclave at 121° C. for 20minutes. The resin was allowed to cool and was used to start theScale-up 1 preparation of resin inoculum. Note: Pasteurized Kymene E7219(using conditions described above) has also been used successful tostart the Scale-up 1 preparation of resin inoculum. Biodehalogenation:Preparation of resin inoculum [Scale-up 1 (SU1)]: A 250-mL round-bottomflask was fitted with a condenser, a pH meter, a temperature controlledcirculating bath, an air sparge tube and a mechanical stirrer. To theflask was added 200 g of sterilized Kymene E7219 and the pH was raisedto 7.2 with 3.28 g of 30% aqueous sodium hydroxide and then 400microliters of HK7 concentrated starter culture was added (1:500, HK7 toresin) [See Example 24 for concentrated starter culture preparation] and1.75 g of a nutrient solution was added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. The pH of the reaction mixture was maintained by periodicaddition of 30% aqueous sodium hydroxide. After 34 hours, 68 microlitersof HK1 concentrated starter culture was added (1:3000, HK1 to resin)[See Example 24 for concentrated starter culture preparation] was added.After 43 hours, the resulting resin was used as inoculum for SU2.

[0270] Scale-up 2 (SU2)

[0271] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 350 g of the pasteurizedresin. The pH was raised to 7.5 with 7.40 g of 30% aqueous sodiumhydroxide and then 5.03 g of Alcalase 2.5L type DX (available fromNovozymes), 87.5 g of the SU1 resin inoculum (20% inoculation rate) and3.06 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. The pH of the reaction mixture was maintained by periodicaddition of 30% aqueous sodium hydroxide. After 23 hours, the resultingresin was used as inoculum for SU3. To increase the molecular weight ofthe resin (as indicated by Gardner-Holdt viscosity or Brookfieldviscosity), a 200 g portion of the remaining resin (Brookfield viscosityof 10 cps) was raised to pH 8.5 with 1.17 g of 30% aqueous sodiumhydroxide and the temperature was raised to 40° C. After 3 hours, theresin had a desirable viscosity and the reaction was quenched by theaddition of concentrated sulfuric acid to pH 2.7. The resulting resinhad a Brookfield viscosity of 25 cps.

[0272] Scale-up 3 (SU3) and General Procedure for repeated Batch Mode

[0273] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 350 g of the pasteurizedresin. The pH was raised to 7.6 with 8.59 g of 30% aqueous sodiumhydroxide and then 4.38 g of Alcalase 2.5L type DX (available fromNovozymes), 87.5 g of the SU2 resin inoculum (20% inoculation rate) and3.06 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. The pH of the reaction mixture was maintained by periodicaddition of 30% aqueous sodium hydroxide. After 23 hours, the resultingresin was used as inoculum for SU4. The pH of the remaining resin wasadjusted to 2.8 with concentrated sulfuric acid and 300 ppm of potassiumsorbate was added as a 10% aqueous solution. This resin had a Brookfieldviscosity of 49 cps. Scale-up 4 (SU4) batch: The procedure was similarto SU3, see Table 6 for data and details.

[0274] Scale-up 5-10 batches: The procedure was similar to SU3 exceptthe 13.40% Kymene E7219 was used without pasteurization (see Table 6 fordata and details). A similar set of experiments with 10% inoculationrate for SU3-SU8 batches resulted in successful, efficient batchbiodehalogenations. TABLE 6 Scale-up 1: 200 g 8% E7219 (autoclaved), noAlcalase, 400 microliters HK7, 1.75 g nutrient solution. Time GardnerOD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH (g)(ppm) (ppm) — 0 7.25 — — 3.28 — — X33031-15-1 1 7.24 — 0.174 — 445 183X33031-15-2 4 7.22 — 0.154 — 402 211 X33031-15-3 20 7.15 — 0.138 — 259309 X33031-15-4 24 7.12 — 0.152 — 222 333 X33031-15-5 28 7.07-7.27 —0.177 0.12 140 375 — 32 7.17 — 0.211 — — — — 34 7.13 — 0.249 Added 68microliters of HK1 inoculum — — 7.13 — 0.272 — — — X33031-15-6 43 6.84 —0.657 — ND 0.36 Scale-up 2: 350 g 13.5% E7219 (pasteurized), 5.03gAlcalase, 87.5g of −15, 3.06 g nutrient solution. Time (pH 7.50) GardnerOD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH (g)(ppm) (ppm) — 0 7.49 — — 7.40 — — X33031-18-1 1 7.39-7.64 A-B 0.128 0.65434 485 X33031-18-2 4 7.41-7.64 A 0.170 0.49 10 814 X33031-18-3 77.50-7.6 A 0.257 0.26 ND 720 X33031-18-4 10 7.44-7.64 A 0.372 0.29 ND380 X33031-18-5 13 7.47-7.59 A 0.543 0.22 ND 0.56 X33031-18-6 23 7.56A-1/A 0.678 — ND 0.38 Inoculum was removed for next batch, the remainderwas set aside for crosslinking. Brookfield viscosity was 10 cps (at pH7.50), crosslinking of 200 g of -18 started 8 hours later: 7.20 — (24 C)7.11 — (31 C) 0 8.50 — 1.17 (31 C) 0.5 8.23 (40 C) 1 8.01 1.5 7.93 A-1/A2 7.82 2.5 7.74 A-B X33047-27-1 3 7.68 Kill reaction with sulfuric acid2.65 Scale-up 3: 350 g 13.5% E7219 (pasteurized), 4.38 g Alcalase, 87.5g of −18, 3.06 g nutrient solution. Time (pH 7.60) Gardner OD₆₀₀ 30% DCPCPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH (g) (ppm) (ppm) — 07.63 — — 8.59 — — X33031-20-1 1 7.51-7.79 A-B 0.148 0.70 476 430X33031-20-2 4 7.54-7.73 A 0.209 0.40 186 616 X33031-20-3 7 7.49-7.74 A0.307 0.56 ND 715 X33031-20-4 10 7.52-7.71 A 0.436 0.37 ND 437X33031-20-5 13 7.49-7.70 A 0.594 0.37 ND 152 X33031-20-6 23 7.48 B-C0.726 — ND 0.29 Scale-up 4: 350 g 13.5% E7219 (pasteurized), 4.38 gAlcalase, 87.5 g of −20, 3.06 g nutrient solution. Time (pH 7.50)Gardner OD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH(g) (ppm) (ppm) — 0 7.60 — — 7.51 — — X33031-23-1 1 7.53-7.62 A-B 0.1760.24 588 360 X33031-23-2 4 7.40-7.61 A-B 0.211 0.48 516 470 X33031-23-37 7.46-7.63 A 0.258 0.38 227 736 X33031-23-4 10 7.47-7.62 A 0.312 0.29ND 811 X33031-23-5 12.5 7.52-7.42 A 0.344 0.24 ND 747 X33031-23-6 257.12 C 0.642 — ND 0.06 Scale-up 5: 350 g 13.5% E7219 (not pasteurized),4.38 g Alcalase, 87.5 g of −23, 3.06 g nutrient solution. Time (pH 7.50)Gardner OD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH(g) (ppm) (ppm) — 0 7.63 — — 6.88 — — X33031-25-1 1 7.37-7.64 B 0.1750.67 499 426 X33031-25-2 3 7.47-7.64 B 0.248 0.39 384 527 X33031-25-3 77.35-7.65 B 0.378 0.71 48 631 X33031-25-4 24 7.12 F-G 0.743 — ND 0.06Scale-up 6: 350 g 13.5% E7219 (not pasteurized), 4.38 g Alcalase, 118 gof −25, 3.06 g nutrient solution. Time (pH 7.3) Gardner OD₆₀₀ 30% DCPCPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH (g) (ppm) (ppm) — 07.41 — — 6.60 — — X33031-27-1 1 7.34-7.51 B-C 0.234 0.43 466 438X33031-27-2 7 7.13-7.33 B-C 0.342 0.42 ND 766 X33031-27-3 19 6.91 B-C0.542 — ND 7.1 Scale-up 7: 350 g 13.5% E7219 (not pasteurized), 4.38 gAlcalase, 87.5 g of −27, 3.06 g nutrient solution. Time (pH 7.35)Gardner OD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH(g) (ppm) (ppm) — 0 7.44 — — 7.27 — — X33031-29-1 1 7.35-7.54 B-C 0.1460.52 676 548 X33031-29-2 4 7.30-7.55 B 0.213 0.70 319 959 X33031-29-3 87.37 A-B 0.301 0.00 ND 1074 X33031-29-4 11 7.29-7.42 A 0.354 0.26 ND 774X33031-29-5 14 7.27-7.43 — 0.402 0.30 ND 442 15 7.33 B — X33031-29-6 237.13 C 0.645 ND 0.13 Acid Test <0.10 2.4 Scale-up 8: 350 g 13.5% E7219(not pasteurized), 4.38 g Alcalase, 87.5 g of −29, 3.06 g nutrientsolution. Time (pH 7.30) Gardner OD₆₀₀ 30% DCP CPD Sample (hours) pH (30C) Viscosity (abs.) NaOH (g) (ppm) (ppm) — 0 7.44 — — 7.17 — —X33031-31-1 1 7.37-7.46 B-C 0.173 0.27 652 504 X33031-31-2 4 7.23-7.34 B0.264 0.28 351 754 X33031-31-3 7 7.16-7.38 A-B 0.361 0.46 ND 921X33031-31-4 10 7.20-7.42 B 0.456 0.47 ND 486 X33031-31-5 13 7.20-7.39 B0.628 0.43 ND 4.6 X33031-31-6 23 7.23 B-C 0.726 ND 0.14 Scale-up 9: 700g 13.5% E7219 (not pasteurized), 8.76 g Alcalase, 175 g of −31, 6.12 gnutrient solution. Time (pH 7.25) Gardner OD₆₀₀ 30% DCP CPD Sample(hours) pH (30 C) Viscosity (abs.) NaOH (g) (ppm) (ppm) — 0 7.27 — —12.86 — — X33031-33-1 1 7.18-7.39 B-C 0.191 1.02 527 402 X33031-33-2 47.15-7.38 B 0.271 0.99 283 672 X33031-33-3 7 7.18-7.40 A-B 0.337 1.05 ND859 X33031-33-4 10 7.24-7.38 A-B 0.397 0.68 ND 619 X33031-33-5 137.27-7.39 B 0.474 0.53 ND 361 X33031-33-6 23 7.11 B-C 0.697 ND 0.12 AcidTest <0.10 2.9 Scale-up 10: 350 g 13.5% E7219 (not pasteurized), 4.38 gAlcalase, 87.5 g of −33, 3.06 g nutrient solution. Time (pH 7.20)Gardner OD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH(g) (ppm) (ppm) — 0 7.17 — — 7.20 — — X33031-35-1 1 7.15-7.34 B-C 0.1880.48 582 462 X33031-35-2 4 7.17-7.32 B 0.293 0.43 299 624 X33031-35-3 77.17-7.31 A-B 0.380 0.45 ND 812 X33031-35-4 10 7.16-7.28 A-B 0.487 0.29ND 487 X33031-35-5 13 7.12-7.30 B 0.647 0.32 ND 150 X33031-35-6 23 7.12A-B 0.820 ND 0.22 Acid Test <0.10 2.7

Examples

[0275] For the following examples TS means total solids

[0276] Demi Water means demineralized water

[0277] DO means dissolved oxygen

Example 23

[0278] Demonstrate feasibility of applying biodehalogenation technologyfor creping aid Crepetrol® 80E with increasing % TS to reduce both1,3-DCP and/or 3-CPD levels to concentrations below 1 ppm.

[0279] Crepetrol® 80E (=A3025) creping aid resin (26.6% TS as received),a tertiary amine-based resin available from Hercules Incorporated(Wilmington, Del.), was obtained from the Voreppe plant, France.

[0280] Three sterile 250 ml Erlenmeyer flasks were charged with 50 mlbatches of resin with increasing % TS (table 7). Dilutions of the resinwere made with sterilized demineralized water. TABLE 7 EPI residuals andtotal solids of Crepetrol ® 80E. Crepetrol ® 80E Demi water EPI 1,3-DCP2,3-DCP 3-CPD TS Sample (ml) (ml) (ppm) (ppm) (ppm) (ppm) (%) 26.6% (asreceived) 50 0 nd 35 nd 100 26.58 20% 37.6 12.4 nd 26 nd 75 20.0 15%28.2 21.8 nd 20 nd 56 15.0

[0281] Prior inoculation, each diluted 50 ml resin was supplemented with0.5 ml nutrient solution and the pH of the solution was adjusted to pH5.8 using a 33% NaOH solution. This nutrient solution contained thefollowing components per L sterilized deminerilized water: 33 g Urea, 5g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 g CaCl₂.2H₂O. A 1 ml concentrated starterculture of both Arthrobacter histidinolovorans (HK1) and Agrobacteriumradiobacter (HK7) was removed from the −80° C. freezer and thawed in awaterbath for 1-2 min. at 30° C. An 50 μl aliquot of the A.histidinolovorans (HK1) suspension and 200 μl aliquot of the A.radiobacter (HK7) suspension were both used to inoculate a 250 mlsterile Erlenmeyer shake flask containing the described dilutions of 50ml supplemented Crepetrol® 80E. After inoculation, the cultures wereincubated for 48 hours at 30° C. in a rotary-shaking incubator (250 rpm;G25 model; New Brunswick Scientific Co., Inc. N.J., USA). Bacterialgrowth was followed in time and determined by measuring the opticaldensity at a wavelength of 600 nm using an Ultrospec 1000 UV/Visspectrophotometer (Pharmacia Biotech, Sweden) and a 3 ml disposablecuvet with 1-cm pathlength (table 8). Samples were pH adjusted to pH 3.5using concentrated sulfuric acid and 0. 1% Proxel® BD (available formZeneca Biocides) was added. Samples were tested for EPI residuals(1,3-DCP and 3-CPD) analysis by GC (table 8). TABLE 8 Bacterial growthin Crepetrol ® 80E with increasing % TS. OD₆₀₀ value C80E 15% C80E 20%C80E 26.6% Time (hrs) TS TS TS 0 0.296 0.256 0.246 16.5 0.558 0.5250.440 19.5 0.570 0.530 0.460 25 0.575 0.545 0.475 41 0.643 0.590 0.49048 0.735 0.600 0.505 1,3-DCP (ppm) after 42 <1 <1 <1 hrs 3-CPD (ppm)after 42 <1 <1 <1 hrs

Example 24 Sequential Enzyme- and Bio-process with High % TS

[0282] Demonstrate efficiency of process started with 3-CPD release viaALCALASE treatment of Crepetrol® 80E, followed by biodehalogenation.Test biodehalogenated product for residual polymer bound 3-CPD using theacid test.

[0283] A. Treatment of 2.5L Crepetrol® 80E

[0284] A clean and sterile 2.5L bioreactor (BioFlo3000 bioreactor,controlled via AFS-BioCommand software; New Brunswick Scientific Co.,Inc. New Jersey, USA) was charged with 2.5 kg Crepetrol® 80E resin(26.6% TS) obtained from Hercules Voreppe plant, France. The pH ofCrepetrol® 80E was adjusted to pH 7.5 using a 33% NaOH solution and thepH-PID controller (Proportional Intergral Display).of the installedbioreactor. Enzyme treatment was started via addition of 12.5 gAlcalase® 2.5L DX (Novozymes). The resin was enzyme treated for a 6 hrstime period using the following incubation conditions:

[0285] pH 7.5

[0286] Temperature controlled at 25° C.

[0287] Agitation controlled at 600 rpm

[0288] Samples (25 ml) were taken in time after 2, 4 and 6 hours tomonitor epi residuals (table 9). Collected samples were pH adjusted topH 3.5 with concentrated sulphuric acid and stored at 4° C. for furtheranalysis. The EPI residuals (3-CPD and the 1,3-DCP ) were analyzed byGC. TABLE 9 3-CPD release in time of treated Crepetrol ® 80E at 26.6%TS. Incubation Sample Time 3-CPD (ppm) 1,3-DCP (ppm) Crepetrol ® 80E 0100 35 26.6% C80E-Alc1 A 2 129 37 C80E-Alc1 B 4 170 39 C80E-Alc1 C 6 16839

[0289] B. Preparation of Pre-cultures of HK1 and HK7 to StartBiodehalogenation Process

[0290] A single colony of A. histidinolovorans (HK1) and a single colonyof A. radiobacter (HK7) (both separately grown on minimal medium saltsmedium containing DCP/CPD) were used to inoculate each separately asterile Erlenmeyer shake flask (250 ml) containing 50 ml of sterileBrain Heart Infusion medium (BHI; Oxoid Ltd, Basingstoke, Hampshire,England; ready made medium, cat.no. CM225). Both pre-cultures wereseparately incubated for 24 h at 30° C. in a temperature controlledrotary-shaking incubator (250 rpm; G25 model; New Brunswick ScientificCo., Inc. New Jersey, USA). The optical density of the batch grown HK1and HK7 culture was determined using an Ultrospec1000 UV/Visspectrophotometer (Pharmacia Biotech, Sweden) at a wavelength of 600 nmand using a 3-ml disposable cuvet with 1-cm pathlength. The growth wasdetermined by measuring the optical density at a wavelength of 600 nmusing a 20-times (water) diluted culture sample (table 10). Thesepre-cultures were used to start the biodehalogenation process of enzymetreated Crepetrol® 80E. TABLE 10 Optical density of 24 hours BHI grownHK1 and HK7 batch culture. OD_(600 nm) (20* Culture dilution) A.histidinolovorans 0.386 HK1 A. radiobacter HK7 0.455

[0291] To make concentrated starter cultures the precultures wereconcentrated via centrifugation (10,000 rpm for 10 min. at 4° C.) andsupplemented with 10% glycerol and then storaged at −80° C.

[0292] C. Biodehalogenation of treated Crepetrol® 80E

[0293] After 6 hrs enzyme treatment (section A), the pH of the resin inthe bioreactor was adjusted to pH 5.8 with concentrated sulphuric acid.The reactor content was supplemented with 25 ml nutrient solution and0.04% PPG2000 (antifoam) (polypropylene glycol P2000 (Fluka Chemie AG,Germany)). This nutrient solution contained the following components perL sterilized demi water: 33 g Urea, 5 g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 gCaCl₂. 2H20. O. Pre-cultures of A. histidinolovorans (HK1) and A.radiobacter (HK7) (50 ml each; section B) were used to start thebiodehalogenation process of the enzyme treated resin. Both culture weresimultaneously used to inoculate a batch fermentation in the 2.5Lbioreactor. Parameter settings of the bioreactor control unit, operatedin batch mode, were as follows:

[0294] pH controlled at pH 5.8 (PID controlled addition of 25% NaOHsolution)

[0295] Temperature controlled at 30° C.

[0296] Stirrer speed 600 rpm (maximum speed of 800 rpm; via DO valuecontrolled)

[0297] Aeration set at 1.0 vvm (2.5L/min; compressed air), minimal DOvalue set at 5% air saturation

[0298] Complete removal of epi residuals from the bioreactor content wasclosely monitored in time, via analysis by gas chromatography (GC-XSD;table 11). After a total incubation time of 51 hours, the batch culturewas finished. The pH of the enzyme treated and biodehalogenated resinwas adjusted to pH 3.5 using concentrated sulphuric acid and the productwas supplemented with 0.2% potassium sorbate and 0.12% Proxel BD. Asample of finished product was used in an acid test to determine thepolymer bound 3-CPD fraction. The pH of this sample (25 ml) was adjustedto pH 1.0 with concentrated sulphuric acid, subsequently the sample wasincubated for 24 hours at 50° C. After incubation the pH was re-adjustedto pH 3.5 with a 33% NaOH solution. Epi residuals were determined viaGC-XSD measurement (table 11). TABLE 11 Epi residuals after sequentialenzyme treatment, bio-treatment and acid test. Process Time 1,3-DCP3-CPD Treatment (hrs) (ppm) (ppm) Crepetrol ® 80E 26.6% 0 35 100 TS1^(st) treatment phase 2 37 129 4 39 170 6 39 168 2^(nd)Biodehalogenation 7 39 168 phase 29.5 nd <1 50 nd <1 Acid Test of C80Efeed 38 188 (Control) Acid Test of Product nd 29

Example 25 Combined Enzyme-Bio-process with high % TS

[0299] Demonstrate efficiency of process with simultaneously started3-CPD release via treatment of Crepetrol® 80E and at the same timebiodehalogenation of free 3-CPD. Test biodehalogenated product forresidual polymer bound 3-CPD using the acid test.

[0300] A clean and sterile 2.5L bioreactor (BioFlo3000 bioreactor,controlled via AFS-BioCommand software; New Brunswick Scientific Co.,Inc. New Jersey, USA) was charged with 2.5 kg Crepetrol® 80E resin(26.6% TS) obtained from Hercules Voreppe plant, France. The resin waspH adjusted to pH 7.5 with a concentrated NaOH (33%) solution,supplemented with 25 ml nutrient solution and 0.04% PPG2000 (antifoam).The nutrient solution contained the following components per Lsterilized demi water: 33 g Urea, 5 g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 gCaCl₂.2H2O. Aliquots of concentrated starter cultures of A.histidinolovorans (HK1) and A. radiobacter (HK7) were removed from the−80° C. freezer and thawed in a waterbath for 1-2 min. at 30° C. Tostart simultaneously the enzyme and biodehalogenation process, thefollowing enzyme/bacteria amounts were added to the supplemented resin:

[0301] 12.5 g Alcalase® 2.5L DX (Novozymes)

[0302] 0.83 ml A. histidinolovorans (HK1) starter culture

[0303] 4.17 ml A. radiobacter (HK7) starter culture

[0304] Parameters settings for the bioreactor control unit, operated inbatch mode, were initially set for the “enzyme treatment phase” asfollows:

[0305] pH controlled at pH 7.5 (PID controlled addition of 25% NaOHsolution)

[0306] Temperature controlled at 25° C.

[0307] Stirrer speed 600 rpm (maximum speed of 800 rpm; via DO valuecontrolled)

[0308] Aeration set at 1.0 vvm (2.5L/min; compressed air), minimal DOvalue set at 5% air saturation

[0309] Samples were taken in time after 2, 4 and 6 hours to monitor epiresiduals (table 12). Epi residuals were measured by GC. These samples(25 ml) were pH adjusted to pH 3.5 with concentrated sulphuric acid andstored at 4° C. for further analysis. After 6 hrs incubation the pH ofthe batch was lowered to pH 5.8 with concentrated sulphuric acid and theincubation temperature was raised to 30° C. Parameters settings for thebioreactor control unit, operated in batch mode during the“biodehalogenation treatment phase”, were set as follows:

[0310] pH controlled at pH 5.8 (PID controlled addition of 25% NaOHsolution)

[0311] Temperature controlled at 30° C.

[0312] Stirrer speed 600 rpm (maximum speed of 800 rpm; via DO valuecontrolled)

[0313] Aeration set at 1.0 vvm (2.5L/min; compressed air), minimal DOvalue set at 5% air saturation

[0314] Complete removal of epi residuals from the bioreactor content wasclosely monitored in time, via analysis by gas chromatography (GC-XSD;table 12). After a total incubation time of 52 hours the batch culturewas finished. The pH of the simultaneously enzyme treated andbiodehalogenated resin was adjusted to pH 3.5 using concentratedsulphuric acid and the product was supplemented with 0.2%potassiumsorbate and 0.12% Proxel BD. A sample of finished product wasused in an acid test to determine the polymer bound 3-CPD fraction. ThepH of this sample (25 ml) was adjusted to pH 1.0 with concentratedsulphuric acid, subsequently the sample was incubated for 24 hours at50° C. After incubation the pH was re-adjusted to pH 3.5 with a 33% NaOHsolution. Epi residuals were determined via GC-XSD measurement (table12). TABLE 12 Epi residuals during simultaneous enzyme- andbio-treatment and after acid test. Process Time 1,3-DCP 3-CPD Treatment(hrs) (ppm) (ppm) Crepetrol ® 80E 26.6% TS 0 35 100 “Enzyme”processconditions 2 nd 141 4 nd 135 6 nd 132 “Biodehalogenation” process 24 <12 conditions 31 nd <1 48 nd <1 Acid Test of C80E feed (control) 38 188Acid Test of Product nd 18

Example 26 Biodehalogenation of Crepetrol® 80E Creping Agent (also knownas Crepetrol® A3025)

[0315] Manufacture and preparation issues.

[0316] (1) Prepare a total of 3225L Crepetrol A3025 without preservative(Available from Hercules Incorporated, Wilmington, Del.)

[0317] (2) Cleaning SU2 reactors:

[0318] Complete fill with hot water (90° C.), aeration and agitation on.Add caustic up to pH 11.

[0319] Keep at 90° C. for 30 min.

[0320] Drain hot/caustic reactor content into SU1 vessel.

[0321] (3) Cleaning SU1 vessel:

[0322] Fill completely with hot (90° C.)/caustic water from SU2 reactor.

[0323] Turn aeration and agitation on in SU1 vessel duringcleaning/heating.

[0324] Drain content after 60 min.

[0325] Complete fill with hot water (90° C.) and drain (=2^(nd) rinse)content of vessel.

[0326] Heat treat (steam) vessel outlets, connectors and all tubing usedfor free draining.

[0327] (4) Pasteurization of Crepetrol A3025:

[0328] Fill reactor SU2 with 3225L Crepetrol A3025 (26% TS) and heatfeedstock to 80° C.

[0329] Turn aeration and agitation on in SU2 reactor duringpasteurization procedure.

[0330] Keep feedstock for 15 min. at 80° C.

[0331] Drain 175L (hot) A3025 in pasteurized SU1 vessel.

[0332] Drain 2000L (hot) A3025 in pasteurized storage vessel(s).

[0333] Keep remaining 1050L pasteurized A3025 in SU2 reactor untilfurther usage in SU2.

[0334] Turn aeration and agitation off.

[0335] (5) Use only pasteurized water (15 min. 90° C.) for dilution stepin SU1.

[0336] (6) Add appropriate amounts of K4 nutrients in dry form (seebelow for nutrient amounts).

[0337] (7) Prepare 0.5L sterile glycerol solution (161 gramsglycerol/500 ml; sterilized 15 min. at 121° C.).

[0338] Scale-up 1 (SU1): Preparation of Resin Inoculum

[0339] (1) Clean and pasteurize SU1 vessel (see above).

[0340] (2) Use a 50% pasteurized and diluted feedstock in SU1 vessel:Charge SU1 vessel with 175L pasteurized Crepetrol A3025 (26% TS) and175L pasteurized (15 min. 90° C.) water.

[0341] (3) Start agitation

[0342] (4) Adjust pH in SU1 to pH 5.8±0.2 with 30% sodium hydroxide.

[0343] (5) Start aeration reactor (0.5 vvm)

[0344] (6) Adjust and maintain temperature in SU1 at 30±1° C.

[0345] (7) Add K4 nutrient in dry form (via a clean container):Component Concentration (g/L) 350L volume Urea 0.33 115.5 grams KH₂PO₄0.10  35.0 grams

[0346] (8) Add 0.5L (161 g/500 ml) sterile glycerol solution (460 ppmfinal conc.).

[0347] (9) Inoculate SU1 with both HK1 and HK7 starter (Appliedinoculation density for HK1 1:3500 and HK7 1:700, inoculum:resin ratio).[See Example 24 for concentrated starter culture preparation.]

[0348] (10) Samples for measurement:

[0349] OD₆₀₀ every 2 hrs

[0350] DCP/CPD values at end SU1

[0351] (11) Incubate for 16-24 hrs at 30±1° C. and pH 5.8±0.2 (Whennecessary correct for pH increases).

[0352] (12) Transfer to SU2 when:

[0353] OD₆₀₀<0.5 or OD₆₀₀ values started to plateau

[0354] Scale-up 2

[0355] (1) Clean and pasteurize SU2 reactor (see above).

[0356] (2) Use 1050L pasteurized Crepetrol A3025 feedstock.

[0357] (3) Start agitation.

[0358] (4) Adjust pH in SU2 to pH 5.8±0.2 with 30% sodium hydroxide.

[0359] (5) Start aeration reactor (0.5 vvm).

[0360] (6) Adjust and maintain temperature in SU2 at 30±1° C.

[0361] (7) Add K4 nutrient in dry form (via a clean container):Component Concentration (g/L) 1050L volume Urea 0.33 346.5 grams KH₂PO₄0.10 105.0 grams

[0362] (8) Inoculate SU2 with 350L SU1 culture (inoculation density 25%)by gravity using a cleaned & pasteurized connector/tubing (see above).

[0363] (9) Samples for measurement:

[0364] OD₆₀₀ every 2 hrs.

[0365] DCP/CPD values at end of SU2.

[0366] (10) Incubate for 16-24 hrs at 30±1° C. and pH 5.8±0.2 (Whennecessary correct for pH increases).

[0367] (11) Start SU3 when:

[0368] DCP/CPD values<5 ppm or when Dissolved Oxygen level increases.

[0369] Incubation time>24 hrs.

[0370] Scale-up 3

[0371] (1) “Pasteurized feedstock” in storage vessel(s):

[0372] Heat-treat (with steam) all equipment used for mixing offeedstock.

[0373] Adjust pH to pH 5.8±0.2 with 30% sodium hydroxide.

[0374] (2) Drain storage vessel(s) by gravity into SU2 reactor (viacleaned/pasteurized connector/tubing (see above)

[0375] (3) Increase aeration volume in accordance with increased volume(0.5 vvm)

[0376] (4) Control agitation and temperature (30° C. ±1° C.) at setvalues.

[0377] (5) Add K4 nutrient in dry form (via clean container) for 2000Lvolume increase: Component Concentration (g/L) 2000L volume Urea 0.33660 grams KH₂PO₄ 0.10 200 grams

[0378] (6) Samples for measurement:

[0379] OD₆₀₀ every 2 hrs.

[0380] DCP/CPD values every 4 hrs.

[0381] Sample for acid test at end of SU3.

[0382] (7) Incubate for 16-24 hrs at 30±1° C. and pH 5.8±0.2 (Whennecessary correct for pH increases)

[0383] (8) Biodehalogenation process in SU3 completed when:

[0384] Total level of [DCP]+[CPD]<5 ppm.

[0385] (9) Product Finishing:

[0386] Adjust pH with concentrated sulfuric acid to pH 3.0±0.2

[0387] Add 2000 ppm (0.2%) potassium sorbate Drain finished productthrough 50-100 μm filter into fresh tote bins.

[0388] Results: EPI—Residuals Analysis TABLE 13 Results Epi-residualdetermination by GC-FID. EPI 1,3-DCP 2,3-DCP 3-CPD Sample [ppm] [ppm][ppm] [ppm] Crepetrol 80E feedstock ND 42 ND 131 This example ND ND NDND Crepetrol 80E feedstock ND 38 ND 227 after acid test This exampleafter acid ND ND ND  56 test

Example 27 Biodehalogenation of Kymene® SLX2 with increasing % TS

[0389] A clean and sterile 500 ml flask was charged with 380 g Kymene®SLX2 (25.3% TS) obtained from Hercules Zwijndrecht plant, TheNetherlands. The pH of the resin was adjusted to pH 5.8 by gradualaddition (while vigorous mixing) of 8.3 g 33% NaOH solution. A series ofsterilized 250 ml Erlenmeyer flasks was charged with 50 ml batches ofresin with increasing % TS. Dilutions of the resin were made usingsterilized deminerilized water (table 14). TABLE 14 Kymene ® SLX2dilution range. Kymene ® SLX2 Demi water Sample (% TS) (ml) (ml) 8 15.834.2 10 19.8 30.2 12 23.7 26.3 14 27.7 22.3 16 31.6 18.4 18 35.6 14.4 2039.5 10.5 25.3 50 0

[0390] Prior inoculation, each diluted resin sample was supplementedwith 0.5 ml filter sterilized nutrient solution. The nutrient solutioncontained the following components per L sterilized demi water: 33 gUrea, 5 g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 g CaCl₂. 2H2O. A 1 ml sample ofconcentrated starter cultures of both A. histidinolovorans (HK1) and A.radiobacter (HK7) was removed from the −80° C. freezer and thawed in awaterbath for 1-2 min. at 30°0 C. An 20 μl aliquot of the A.histidinolovorans (HK1) suspension and 100 μl aliquot of the A.radiobacter (HK7) suspension were both used to inoculate the described50 ml dilutions of supplemented Kymene® SLX2. After inoculation, thecultures were incubated for 22 hours at 30° C. in a rotary-shakingincubator (250 rpm; G25 model; New Brunswick Scientific Co., Inc. NewJersey, USA). Bacterial growth was followed in time and determined bymeasuring the optical density at a wavelength of 600 nm using anUltrospec1000 UV/Vis spectrophotometer (Pharmacia Biotech, Sweden) and a3 ml disposable cuvet with 1-cm pathlength (table 8). Samples were pHadjusted to pH 2.8 using concentrated sulfuric acid and 0.1% Proxel BDwas added. Samples were measured for EPI residuals (3-CPD and 1,3-DCP)analysis by GC (table 15). TABLE 15 Bacterial growth in Kymene ® SLX2with increasing % TS. t = 22 hrs incubation Sample OD₆₀₀ nm 3-CPD1,3-DCP “Viscosity” (% TS) 0 hrs 17 hrs 22 hrs (ppm) (ppm) appearance 80.141 0.655 0.654 <10 <10 − 10 0.126 0.742 0.717 <10 <10 − 12 0.1220.797 0.762 <10 <10 − 14 0.114 0.705 0.769 <10  32 − 16 0.107 0.6090.623 <10 294 + 18 0.094 0.556 0.560 <10 407 ++ 20 0.096 0.486 0.541 <10492 ++ 25.3 0.095 0.139 0.130 gelled gelled +++

Example 28 Biodehalogenation of Kymene® E7220 (Acid Treated Material)with Increasing % TS

[0391] A clean and sterile 500 ml flask was charged with 300 g Kymene®E7220 (22.5% TS) obtained from Hercules Voreppe plant, France. The pH ofthe resin was adjusted to pH 7.0 by gradual addition (while vigorousmixing) of 15.3 g 33% NaOH solution. A series of sterilized 250 mlErlenmeyer flasks was charged with 50 ml batches of resin withincreasing % TS. Dilutions of the resin were made using sterilizeddemineralized water (table 16). TABLE 16 Kymene ® E7220 dilution range.Kymene ® E7220 Demi water Sample (% TS) (ml) (ml) 8 17.8 32.2 10 22.227.8 12 26.7 23.3 14 31.1 18.9 16 35.6 14.4 18 40.0 10.0 20 44.4 5.622.5 50 0

[0392] Prior inoculation, each diluted resin sample was supplementedwith 0.5 ml filter sterilized nutrient solution. The nutrient solutioncontained the following components per L sterilized demi water: 33 gUrea, 5 g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 g CaCl₂.2H2O. A 1 ml sample ofconcentrated starter cultures of both A. histidinolovorans (HK1) and A.radiobacter (HK7) was removed from the −80° C. freezer and thawed in awaterbath for 1-2 min. at 30° C. An 20 μl aliquot of the A.histidinolovorans (HK1) suspension and 100 μl aliquot of the A.radiobacter (HK7) suspension were both used to inoculate the described50 ml dilutions of supplemented Kymene® E7220. After inoculation (startOD₆₀₀=0.208), the cultures were incubated for 91 hours at 30° C. in arotary-shaking incubator (250 rpm; G25 model; New Brunswick ScientificCo., Inc. New Jersey, USA). Bacterial growth was followed in time anddetermined by measuring the optical density at a wavelength of 600 nmusing an Ultrospec1000 UV/Vis spectrophotometer (Pharmacia Biotech,Sweden) and a 3 ml disposable cuvet with 1-cm pathlength (table 17).Samples were pH adjusted to pH 2.8 using concentrated sulfuric acid and0.1% Proxel BD was added. Samples were measured for EPI residuals (3-CPDand 1,3-DCP) by GC analysis (table 17). TABLE 17 Bacterial growth inKymene ® E7220 with increasing % TS. OD₆₀₀ nm Sample (% 19 27 91 t = 91hrs incubation “Viscosity” TS) hrs hrs hrs 3-CPD (ppm) 1,3-DCP (ppm)appearance 8 1.10 1.073 nd <10 <10 − 3 10 1.19 1.168 nd <10 <10 − 0 121.14 1.205 nd <10 <10 − 5 14 0.87 1.237 nd <10 <10 − 0 16 0.50 1.1971.025 <10 <10 − 0 18 0.27 0.647 1.095 <10 <10 − 6 20 0.19 0.290 1.176<10 <10 − 5 25.3 0.13 0.140 1.067 <10  49 − 9

Example 29 Biodehalogenation of Kymene® 736 (Polyamine/Azetidinium basedResin) at 15-20% TS in 50 ml Batch

[0393] Kymene® 736 (Crepetrol® 73) creping aid resin (39.6% TS asreceived), a polyamine/azetidinium-based resin available from HerculesIncorporated (Wilmington, Del.), was obtained from the Voreppe plant,France.

[0394] A clean and sterile 250 ml flask was charged with 100 g Kymene®736 (39.6% TS) and the pH of the resin was adjusted to pH 7.0 by gradualaddition (while vigorous mixing) of a 33% NaOH solution. Two sterile 250ml Erlenmeyer flasks were charged with a 50 ml batch of resin dilutedeither to 15% or 20% TS. Dilutions of the resin were made usingsterilized deminerilized water (table 18). TABLE 18 Kymene ® 736dilution range. Kymene ® 736 Demi water Sample (% TS) (ml) (ml) 15 19.031.0 20 25.2 24.8

[0395] Prior inoculation, each diluted resin sample was supplementedwith 0.5 ml filter sterilized nutrient solution. The nutrient solutioncontained the following components per L sterilized demi water: 33 gUrea, 5 g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 g CaCl₂. 2H2O. A 1 ml sample ofconcentrated starter cultures of both A. histidinolovorans (HK1) and A.radiobacter (HK7) was removed from the −80° C. freezer and thawed in awaterbath for 1-2 min. at 30° C. An 50 μl aliquot of the A.histidinolovorans (HK1) suspension and 200 μl aliquot of the A.radiobacter (HK7) suspension were both used to inoculate the described50 ml dilutions of supplemented Kymene® 736. After inoculation, thecultures were incubated for 43 hours at 30° C. in a rotary-shakingincubator (250 rpm; G25 model; New Brunswick Scientific Co., Inc. NewJersey, USA). Bacterial growth was followed in time and determined bymeasuring the optical density at a wavelength of 600 nm using anUltrospec1000 UV/Vis spectrophotometer (Pharmacia Biotech, Sweden) and a3 ml disposable cuvet with 1-cm pathlength (table 19). Samples were pHadjusted to pH 2.8 using concentrated sulfuric acid and 0.1% Proxel BDwas added. TABLE 19 Bacterial growth in Kymene ® 736 with increasing %TS. Time OD_(600 nm) (hrs) 15% TS 20% TS 0 0.441 0.378 20 0.935 0.419 231.081 0.430 41 0.997 0.910 43 nd 0.899

Example 30 Biodehalogenation of Kymene® 736 at 20% TS in 2L Batch

[0396] Kymene® 736 (Crepetrol® 73) creping aid resin (39.6% TS asreceived), a polyamine/azetidinium-based resin available from HerculesIncorporated (Wilmington, Del.), was obtained from the Voreppe plant,France.

[0397] A clean and sterile 2.5L bioreactor (BioFlo3000 bioreactor,controlled via AFS-BioCommand software; New Brunswick Scientific Co.,Inc. New Jersey, USA) was charged with 1010 ml Kymene® 736 resin (39.6%TS) and 990 ml sterile demineralized water. The diluted (20% TS) resinwas pH adjusted to pH 7.0 with a concentrated NaOH (33%) solution,supplemented with 20 ml nutrient solution and 0.0025% PPG2000(antifoam). The nutrient solution contained the following components perL sterilized demi water: 33 g Urea, 5 g KH₂PO₄, 5 g MgSO₄.7H₂O and 1 gCaCl₂.2H2O. Aliquots of concentrated starter cultures of A.histidinolovorans (HK1) and A. radiobacter (HK7) were removed from the−80° C. freezer and thawed in a waterbath for 1-2 min. at 30° C. A 2 mlsample of the A. histidinolovorans (HK1) suspension and 8 ml sample ofthe A. radiobacter (HK7) suspension was used to inoculate simultaneouslythe content of the 2.5L bioreactor. Parameter settings of the bioreactorcontrol unit, operated in batch mode, were as follows:

[0398] pH controlled at pH 7.0 (PID controlled addition of 25% NaOHsolution)

[0399] Temperature controlled at 30° C.

[0400] Stirrer speed 600 rpm (maximum speed of 800 rpm; via DO valuecontrolled)

[0401] Aeration set at 1.0 vvm (2.0L/min; compressed air), minimal DOvalue set at 5% air saturation

[0402] Complete removal of epi residuals from the bioreactor content wasmonitored in time, via analysis by gas chromatography (GC-XSD; table20). After a total incubation time of 48 hours, the batch culture wasfinished. The pH of the biodehalogenated resin was adjusted to pH 2.8using concentrated sulphuric acid and the resin was supplemented with0.02% potassiumsorbate and 0.12% Proxel BD. TABLE 20 Epi residuals andbacterial growth in Kymene ® 736 at 20% TS. 1,3-DCP Time (hrs)OD_(600 nm) (ppm) 3-CPD (ppm) 0 0.412 — — 5 0.422 — — 22 0.572 — — 270.697 — — 30 0.818 <1 18 46 0.840 <1 <1

Example 31 Alcalase® Treatment of Tertiary Amine Based Resin

[0403] The following procedure has been used to promote 3-MCPD formationvia hydrolysis of the polymer-bound chlorohydrin species in Crepetrol®A3025 (Hercules Incorporated, Wilmington, Del.).

[0404] A sample of 191.88 g of Crepetrol®D A3025, containing no addedpreservatives was placed in a 250 glass flask, provided with a plastic,sealed screw cap. The weight of the sample was measured with a MettlerLaboratory digital scale with a precision of ±0.005 g. The total solidconcentration of the Crepetrol® A3025 was determined in a separateexperiment measuring the weight loss after 15 min at 150° C. The sampletotal solid concentration was found to be 26.9%.

[0405] The pH was carefully adjusted to 7.00 with a 10% w/w NaOHsolution (total 12.52 g), while stirring with a magnetic stirrer,monitoring the pH with a Mettler pH-meter (MP 220), equipped with anInLab electrode (combination electrode, internal ref. ARGENTHAL with Agion trap). The pH meter was calibrated for the 7.00-10.00 pH range priorto the pH adjustment.

[0406] The sample was placed in an ice-melting bath (0° C.). 0.9 g ofAlcalase® 2.5 L DX (obtained from Novozymes) were added to theCrepetrol®A3025 sample.

[0407] The flask was then placed in a horizontally shaking(200strokes/min) thermostatic bath at 25° C., and left in agitation for14 hours.

[0408] After 14 hours the sample was removed and the pH was adjustedwith conc. H₂SO₄ to 3.5.

[0409] A sample of the original material (Crepetrol® A3025) and a sampleof the material after the above treatment were analysed using agas-chromatograpic technique to measure their content of3-monochloropropandiol.

[0410] The amount of 3-monochloropropandiol produced during thetreatment was calculated as the difference of 3-monochloropropandiolconcentration of the sample after treatment and the original sample ofCrepetrol® A3025.

[0411] The Reduced Viscosity of the final sample was also measured usingan Ubbelohde capillary at 25° C. A 2% solution in 1N Ammonium Chloridewas prepared and the time to flow through the capillary was measured.The time of flow of the Ammonium chloride solution was measured as well.Reduced viscosity was calculated according to the equation:

RSV[dl/g]={(time_(sample)[sec]/time_(solvent)[sec])−1}/Conc_(sample)[g/100 ml]

[0412] The results for CPD release were the following:

Released 3-MCPD[ppm]=Conc. _(after treatment)[ppm]−Conc._(initial)[ppm]=182−101.9=80.1 ppm

[0413] In the following page is reported a table showing the results ofa series of experiments preformed with this procedure, changing theconditions of enzyme dosage, pH, total solid, temperature and durationof treatment.

[0414] The example given corresponds to number 31-4 of table21. TABLE 21#std Alcalase TS RELEASE delta delta (sample enzyme Enzyme % actual temptime D RATIO delta visc. RSV marking) g weight TS % calc degC. pH hs3-MCPD visc % in cP RSV abs 31-2 0.9 0.45 15 15.0 25 7 6 0.548 −5.8 −1.60.265 −0.001 31-3 0.45 0.225 26 26.0 25 7 6 0.360 −1.6 −1.2 0.265 −0.00131-9 0.47 0.235 15 15.0 25 8 6 0.486 −11.1 −3.1 0.266 0 31-12 0.9 0.4526 26.0 25 8 6 0.964 9.2 6.6 0.274 0.008 31-21 0.675 0.3375 20.5 20.5 257.5 10 0.729 −16.8 −7.6 0.261 −0.005 31-1 0.45 0.225 15 15.0 25 7 140.568 3.9 1.1 0.265 −0.001 31-11 0.45 0.2116 26 24.4 25 8 14 0.852 49.431.4 0.354 0.088 31-4 0.9 0.4384 26 25.3 25 7 14 0.974 −0.5 −0.4 0.266 031-10 0.9 0.45 15 15.0 25 8 14 0.938 −4.7 −1.3 0.27 0.004 31-25 0.6750.3372 20.5 20.5 32.5 7.5 2 0.325 −5.4 −2.4 0.264 −0.002 31-27 0.730.365 20.5 20.5 32.5 7.5 10 0.639 −7.5 −3.4 0.265 −0.001 31-29 0.6750.3375 20.5 20.5 32.5 7.5 10 0.519 −16.8 −7.6 0.267 0.001 31-23 0.6750.3375 20.5 20.5 32.5 6.5 10 0.218 −14.8 −6.7 0.267 0.001 31-20 0.6750.329 26 25.3 32.5 7.5 10 0.645 −1.6 −1.1 0.28 0.014 31-24 0.675 0.337520.5 20.5 32.5 8.5 10 0.579 81.0 36.5 0.453 0.187 31-30 0.675 0.337420.5 20.5 32.5 7.5 10 0.639 −8.8 −4.0 0.266 0 31-28 0.675 0.3375 20.520.5 32.5 7.5 10 0.444 −9.5 −4.3 0.27 0.004 31-19 0.675 0.3375 9.5 9.532.5 7.5 10 0.544 −58.6 −10.2 0.27 0.004 31-18 1.13 0.5649 20.5 20.532.5 7.5 10 0.910 −6.2 −2.8 0.262 −0.004 31-17 0.21 0.105 20.5 20.5 32.57.5 10 0.203 13.8 6.2 0.272 0.006 31-26 0.675 0.3375 20.5 20.5 32.5 7.518 0.789 −5.5 −2.5 0.27 0.004 31-15 0.45 0.225 26 26.0 40 8 6 0.017440.8 319.8 0.685 0.419 31-5 0.45 0.225 15 15.0 40 7 6 0.383 −14.3 −4.00.255 −0.011 31-8 0.9 0.45 26 26.0 40 7 6 0.609 4.2 3.0 0.277 0.01131-14 0.9 0.45 15 15.0 40 8 6 0.814 14.2 4.0 0.319 0.053 31-6 0.9 0.4515 15.0 40 7 14 0.938 −12.2 −3.4 0.268 0.002 31-7 0.45 0.225 26 26.0 407 14 0.680 24.0 17.4 0.31 0.044 31-13 0.45 0.225 15 15.0 40 8 14 0.65093.8 26.3 0.53 0.264 31-16 0.9 0.45 26 26.0 40 8 14 na gelled gelled 10.734 31-22 0.675 0.3375 20.5 20.5 47.5 7.5 10 0.189 12.5 5.6 0.306 0.04

[0415] According to the result reported is statistically calculated thatthe best conditions for the enzyme treatment of this resin are: Enzymeconc, [% W]: 0.45 TS polymer [%]: 22.17 Temp [°] 25 pH 7.94 Duration[hs] 10.43

[0416] This treatment will result in a high release of 3-MCPD (ca. 95%)and in no increase in final viscosity. (Higher final viscosity is aproblem for product stability, especially if additional treatment of theresin is required).

[0417] According to the statistical model elaborated, alternativeconditions can be chosen when required resulting in similar finalefficiency. The following conditions for example where an even loweramount of enzyme is used, and a final release of ca.90% of 3-MCPD isexpected with no significant viscosity increase. Enzyme conc, [% W]:0.25 TS polymer [%]: 22.4 Temp [°] 25 pH 8.00 Duration [hs] 14.00

Example 32 Adhesion Measurements Results

[0418] In the following chart are reported the results of the adhesionmeasurement of a selected number of enzyme treated samples (extractedfrom the series reported in the table above). Significant hydrolysis(and consequent drop in average MW) of the polymer can result insignificant peel strength loss, so we wanted to check if any importantdrop in adhesion was detectable.

[0419] Peel test was measured by soaking a strip of fabric in a 2%solids solution of the creping aid and then curing the strip for 7,5minutes at 92° C. in contact with a standard metal plate (mild steel).The average force to peel away the strip from the plate was measuredusing a Zwick005 universal testing machine.

[0420] Results are plotted against the observed viscosity variationafter enzyme treatment. Is clearly visible that the adhesion of thesample is distributed randomly (with an oscillation due to experimentalvariation), independently from the observed change in viscosity.Furthermore the values are all distributed around the typical value forthe untreated material (0.75-0.8 N/cm)

[0421] These results indicate that the enzyme treatments didn't causeany measurable decrease in the adhesion strength of the polymer. TABLE22 peel strength std# N/cm Delta RSV 31-4  0.7 0 31-10 0.65 0.004 31-110.71 0.088 31-12 0.75 0.008 31-21 0.72 −0.005 31-18 0.81 −0.004 31-240.82 0.187 29 0.74 0.001  5 0.76 −0.011  6 0.93 0.002 13 0.69 0.264 140.77 0.053 15 0.74 0.419

Example 33 Biodehalogenation of Crepetrol® 870 (see Table 23 for Dataand Details)

[0422] A portion of Crepetol® 870 without biocide (Available fromHercules Incorporated, Wilmington, Del.; Voreppe, France plant) wasdiluted to 18.9% total solids with deionized water. This diluted resinhad a Brookfield viscosity of 53 cps.

[0423] Pasteurization: A 2-L round-bottom flask was fitted with acondenser, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 1780 g of the 18.9% resin. The resin hada pH of 4.6 and was heated over one hour from 25° C. to 85° C. The resinwas held at 85° C. for 20 minutes and then cooled to 25° C. in 45minutes. The pasteurized resin was stored in a sterile container.

[0424] Biodehalogenation: Preparation of resin inoculum [Scale-up 1(SU1)]: A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. A portion of the pasteurized resin was diluted to10% with sterile, deionized water. To the flask was added 198 g of this10% resin and 2.0 g of 5 mM sterile glycerol in water solution. The pHwas raised to 5.8 with 3.18 g of 30% aqueous sodium hydroxide and then68 microliters of HK1 concentrated starter culture was added (1:3000,HK1 to resin) [See Example 24 for concentrated starter culturepreparation] and 1.75 g of a nutrient solution was added. (The nutrientsolution consisted of 8026 ppm of potassium dihydrogen phosphate, 27480ppm of urea, 4160 ppm of magnesium sulfate and 840 ppm of calciumchloride in tap water.) The air sparge was started, the temperature wasmaintained at 30° C. The bacterial growth was monitored by opticaldensity (OD₆₀₀) and the biodehalogenation was monitored by GC. OD₆₀₀ wasdetermined by measuring the optical density at a wavelength of 600 nmusing a Spectronic® Genesys™ UV/Vis spectrophotometer (SpectronicInstruments, Incorporated, Rochester, N.Y., USA) and a disposable cuvetwith 1-cm pathlength. After 17 hours, the resulting resin was used asinoculum for SU2.

[0425] Scale-up 2 (SU2)

[0426] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurized18.9% resin. The pH was raised to 5.8 with 4.52 g of 30% aqueous sodiumhydroxide and then 50.0 g of the SU1 resin inoculum was added (25%inoculation rate) and 1.31 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 8 hours,the resulting resin was used as inoculum for SU3.

[0427] Scale-up 3 (SU3)

[0428] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurized18.9% resin. The pH was raised to 5.8 with 4.45 g of 30% aqueous sodiumhydroxide and then 50.0 g of the SU2 resin inoculum was added (25%inoculation rate) and 1.31 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 14.5hours, the resulting resin was used as inoculum for SU4. The remainingresin not used for inoculum was discarded, but could have been used togive the finished product.

[0429] Scale-up 4 (SU4)

[0430] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized18.9% resin. The pH was raised to 5.8 with 8.94 g of 30% aqueous sodiumhydroxide and then 100.0 g of the SU3 resin inoculum was added (25%inoculation rate) and 2.62 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 8 hours,the resulting resin was used as inoculum for SU5. The remaining resinnot used for inoculum was discarded, but could have been used to givethe finished product.

[0431] Scale-up 5 (SU5)

[0432] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized18.9% resin. The pH was raised to 5.8 with 8.96 g of 30% aqueous sodiumhydroxide and then 100.0 g of the SU4 resin inoculum was added (25%inoculation rate) and 2.62 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 15.5hours, the resulting resin was used as inoculum for SU6. The remainingresin not used for inoculum was converted to finished product bylowering the pH to 4.7 with 85% phosphoric acid and adding 2000 ppm ofpotassium sorbate as biocide.

[0433] Scale-up 6 (SU6)

[0434] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized18.9% resin. The pH was raised to 5.8 with 8.84 g of 30% aqueous sodiumhydroxide and then 100.0 g of the SU5 resin inoculum was added (25%inoculation rate) and 2.62 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 8 hours,the resulting resin was converted to finished product by lowering the pHto 4.7 with 7.20 g of 85% phosphoric acid and adding 2000 ppm ofpotassium sorbate (7.11 mL of 10 wt % aqueous potassium sorbate) asbiocide. See Table 23 for the results from monitoring the treatment.TABLE 23 Acid tests on Crepetrol 870 DCP CPD Sample (ppm) (ppm)X33047-19A Voreppe before Pasteurization ND 47 X33047-19A After AcidTest ND 71 X33047-39 Voreppe after Pasteurization ND 43 X33047-39 AfterAcid Test ND 69 Scale-up 1: 198 g 10% Crepetrol 870, 2 g 0.5 M glycerol,68 microliters HK1, 1.75 g nutrient solution. Time OD₆₀₀ 30% DCP CPDSample (hours) pH (30 C) (abs.) NaOH (g) (ppm) (ppm) — 0 5.81 — 3.18 ND23 X33047-41-1 1 5.81 0.020 — ND 24 X33047-41-2 17 5.82 0.473 — 0.260.08 Scale-up 2: 150 g 18.87% Crepetrol 870, 50.0 g −41, 1.31 g nutrientsolution. Time OD₆₀₀ 30% DCP CPD Sample (hours) pH (300) (abs.) NaOH (g)(ppm) (ppm) — 0 5.82 — 4.52 ND 32 X33047-43-1 1 5.82 0.126 — ND 28X33047-43-2 4 5.83 0.159 — 1.0  1.12 X33047-43-3 8 5.82 0.168 — 0.570.50 Scale-up 3: 150 g 18.87% Crepetrol 870, 50.0 g −43, 1.31 g nutrientsolution. Time OD₆₀₀ 30% DCP CPD Sample (hours) pH (300) (abs.) NaOH (g)(ppm) (ppm) — 0 5.81 — 4.45 ND 32 X33047-45-1 0.117 5.81 0.048 — ND 29X33047-45-2 14.5 5.81 0.093 — 0.56 0.49 Scale-up 4: 300 g 18.87%Crepetrol 870, 100.0 g −45, 2.62 g nutrient solution. Time OD₆₀₀ 30% DCPCPD Sample (hours) pH (300) (abs.) NaOH (g) (ppm) (ppm) — 0 5.82 — 8.94ND 32 X33047-47-1 0.25 5.82 0.029 — ND 30 X33047-47-2 4.25 5.82 0.067 —0.58 0.30 X33047-47-3 8 5.81 0.065 — 0.59 0.72 Scale-up 5: 300 g 18.87%Crepetrol 870, 100.0 g −47, 2.62 g nutrient solution. Time OD₆₀₀ 30% DCPCPD Sample (hours) pH (300) (abs.) NaOH (g) (ppm) (ppm) — 0 5.81 — 8.96ND 32 X33047-49-1 0.083 5.81 0.053 — ND 33 X33047-49-2 15.5 5.80 0.049 —0.6 0.12 X33047-49 Acid test ND 29 Scale-up 6: 300 g 18.87% Crepetrol870, 100.0 g −49, 2.62 g nutrient solution. Time OD₆₀₀ 30% DCP CPDSample (hours) pH (300) (abs.) NaOH (g) (ppm) (ppm) — 0 5.80 — 8.84 ND32 X33047-51-1 0.083 5.80 0.023 — ND 32 X33047-51-2 4 5.80 0.039 — ND8.7 X33047-51-3 8 5.80 0.050 — 0.59 1.0 X33047-51 Acid test ND 30

Example 34 Adhesion Testing for Creping Agents

[0435] A device for evaluating the adhesive properties of potentialcreping adhesives has been constructed. This apparatus consists of aheated cast iron block that is mounted on the actuator of a MTS testinstrument. This platen is heated to 120° C . A paper sample is attachedto the upper platen of the load cell of the test instrument with doublesided tape. To perform the test, a known quantity of an aqueous solutionof creping adhesive with a known concentration is sprayed onto theheated block. This is accomplished by using an airbrush that has beenfitted with a volumetric spray bottle. The volumetric spray bottleallows one to accurately measure the volume of solution that is to beapplied to the test platen. Our standard test conditions use a volume of1.2 mL of a 4.0% solids aqueous solution. The pH of the solution can beambient or can be adjusted to 7.0 prior to testing. After the resinsolution is sprayed onto the heated block, the actuator is raised tocontact the heated block to the paper sample with a force of 10 kg. Theactuator is then lowered and the force to pull the platen away from thepaper that it has contacted. This measured force is the adhesion valueof the particular resin being tested. Since the applied force is notalways exactly 10 kg the adhesion value is normalized to account forslight variations in the applied force. This is accomplished bymultiplying the measured adhesion value by [10/(Applied force in kg)].The paper used for testing is a 40 lb. basis weight sheet prepared froma 50/50 hardwood/softwood bleached Kraft furnish.

[0436] The following table contains Adhesion test and Brookfieldviscosity data: TABLE 24 Viscosity Test (Kgs) Test (Kgs) Designation(cps) (ambient pH) (pH 7.0) X33047-19A Comp. Ex. 53 23.4 21.7 X33047-39Comp. Ex. 54 23.4 23.2 X33047-47 Example 45 — — X33047-49 Example 4621.6 22.1 X33047-51 Example 49 23.7 22.2

[0437] The data in this table indicate that the present invention hasviscosity and Adhesion Tests comparable to the untreated resins,indicating the performance of the biodehalogenated resins is comparableto the resins that were not biodehalogenated.

Example 35 Alcalase-Biodehalogenation of Crepetrol® 870 (see Table 25for Data and Details)

[0438] A portion of Crepetol® 870 without biocide (Available fromHercules Incorporated, Wilmington, Del.; Voreppe, France plant) wasdiluted to 18.7% total solids with deionized water. This diluted resinhad a Brookfield viscosity of 53 cps.

[0439] Pasteurization: A 2-L round-bottom flask was fitted with acondenser, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 2942 g of the 18.7% resin. The resin hada pH of 4.6 and was heated over 1.5 hour from 25° C. to 85° C. The resinwas held at 85° C. for 20 minutes and then cooled to 25° C. in 30minutes. The pasteurized resin was stored in a sterile container.

[0440] Biodehalogenation: Preparation of resin inoculum [Scale-up 1(SU1)]: A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. A portion of the pasteurized resin was diluted to10% with sterile, deionized water. To the flask was added 198 g of this10% resin and 2.0 g of 5 mM sterile glycerol in water solution. The pHwas raised to 7.2 with 8.31 g of 30% aqueous sodium hydroxide and then68 microliters of HK1 concentrated starter culture was added (1:3000,HK1 to resin) [See Example 24 for concentrated starter culturepreparation] and 1.75 g of a nutrient solution was added. (The nutrientsolution consisted of 8026 ppm of potassium dihydrogen phosphate, 27480ppm of urea, 4160 ppm of magnesium sulfate and 840 ppm of calciumchloride in tap water.) The air sparge was started, the temperature wasmaintained at 30° C. The bacterial growth was monitored by opticaldensity (OD₆₀₀) and the biodehalogenation was monitored by GC. OD₆₀₀ wasdetermined by measuring the optical density at a wavelength of 600 nmusing a Spectronic® Genesys™ UV/Vis spectrophotometer (SpectronicInstruments, Incorporated, Rochester, N.Y., USA) and a disposable cuvetwith 1-cm pathlength. After 16 hours, the resulting resin was used asinoculum for SU2.

[0441] Scale-up 2 (SU2)

[0442] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurized18.7% resin. The pH was raised to 7.2 with 10.97 g of 30% aqueous sodiumhydroxide and then 1.02 g of Alcalase 2.5L type DX (available fromNovozymes), 50.0 g of the SU1 resin inoculum (25% inoculation rate) and1.31 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 8 hours, the resulting resin was used as inoculum forSU3.

[0443] Scale-up 3 (SU3)

[0444] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurized18.7% resin. The pH was raised to 7.2 with 11.42 g of 30% aqueous sodiumhydroxide and then 0.87 g of Alcalase 2.5L type DX (available fromNovozymes), 50.0 g of the SU2 resin inoculum (25% inoculation rate) and1.31 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 14 hours, the resulting resin was used as inoculum forSU4. The remaining resin not used for inoculum was discarded, but couldhave been used to give the finished product.

[0445] Scale-up 4 (SU4)

[0446] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized18.7% resin. The pH was raised to 7.2 with 22.17 g of 30% aqueous sodiumhydroxide and then 1.73 g of Alcalase 2.5L type DX (available fromNovozymes), 100.0 g of the SU3 resin inoculum (25% inoculation rate) and2.62 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 8 hours, the resulting resin was used as inoculum forSU5. The remaining resin not used for inoculum was converted to finishedproduct by lowering the pH to 4.7 with 85% phosphoric acid and adding2000 ppm of potassium sorbate as biocide.

[0447] Scale-up 5 (SU5)

[0448] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized18.7% resin. The pH was raised to 7.2 with 22.77 g of 30% aqueous sodiumhydroxide and then 1.73 g of Alcalase 2.5L type DX (available fromNovozymes), 100.0 g of the SU4 resin inoculum (25% inoculation rate) and2.62 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 14.5 hours, the resulting resin was used as inoculumfor SU6. The remaining resin not used for inoculum was converted tofinished product by lowering the pH to 4.7 with 85% phosphoric acid andadding 2000 ppm of potassium sorbate as biocide.

[0449] Scale-up 6 (SU6)

[0450]

[0451] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized18.7% resin. The pH was raised to 7.2 with 23.02 g of 30% aqueous sodiumhydroxide and then 1.73 g of Alcalase 2.5L type DX (available fromNovozymes), 100.0 g of the SU5 resin inoculum (25% inoculation rate) and2.62 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 8 hours, the resulting resin was converted to finishedproduct by lowering the pH to 4.7 with 22.5 g of 85% phosphoric acid andadding 2000 ppm of potassium sorbate (7.69 mL of 10 wt % aqueouspotassium sorbate) as biocide.

[0452] See Table 25 for the results from monitoring the treatment. Notethat using this inoculation rate, the SU6 batch was not completelybiodehalogenated within an 8 hour reaction time. A side-by-sideexperiment, with the same reaction times, using a 33% inoculation ratein the SU4 batch and a 50% inoculation rate in the SU5 and SU6 batchesprovide a complete biodehalogenation in the SU6 batch (see Table 26)TABLE 25 30% Time OD₆₀₀ NaOH DCP CPD Sample (hours) pH (30 C) (abs.) (g)(ppm) (ppm) Scale-up 1: 198 g 10% Crepetrol 870, 2 g 0.5 M glycerol, 68microliters HK1, 1.75 g nutrient solution. — 0 7.20 —  8.31 ND 23X33047-62-1 0.5 7.20 0.066 — ND 23 X33047-62-2 15.67 7.21 0.180 — ND 3.3Scale-up 2: 150 g 18.73% Crepetrol 870, 50.0 g −62, 1.02 g of Alcalase,1.31 g nutrient solution. — 0 7.18 — 10.97 ND 32 X33047-64-1 1 7.160.180 — ND 34 X33047-64-2 4 7.19 0.199 — ND 11 X33047-64-3 8 7.18 0.253— 0.41 0.48 Scale-up 3: 150 g 18.73% Crepetrol 870, 50.0 g −64, 0.87 gof Alcalase, 1.31 g nutrient solution. — 0 7.21 — 11.24 ND 32X33047-66-1 0.083 7.21 0.207 — ND 33 X33047-66-2 13.75 7.17 0.299 — 0.420.25 Scale-up 4: 300 g 18.73% Crepetrol 870, 100.0 g −66, 1.73 g ofAlcalase, 2.62 g nutrient solution. — 0 7.23 — 22.17 ND 32 X33047-68-1 17.21 0.196 — ND 32 X33047-68-2 4 7.20 0.228 — ND 19 X33047-68-3 8 7.200.260 — 0.47 0.46 X33047-68 After Acid Test 0.37 5.9 Scale-up 5: 300 g18.73% Crepetrol 870, 100.0 g −68, 1.73 g of Alcalase, 2.62 g nutrientsolution. — 0 7.23 — 22.77 ND 32 X33047-70-1 0.083 7.23 0.165 — ND 34X33047-70-2 14.5 7.19 0.247 — 0.43 0.2 X33047-70 After Acid Test 0.352.9 Scale-up 6: 300 g 18.73% Crepetrol 870, 100.0 g −70, 1.73 g ofAlcalase, 2.62 g nutrient solution. — 0 7.24 — 23.02 ND 32 X33047-72-1 17.23 0.195 — ND 33 X33047-72-2 4 7.22 0.208 — ND 35 X33047-72-3 8 7.220.233 — ND 25

[0453] TABLE 26 30% Time OD₆₀₀ NaOH DCP CPD Sample (hours) pH (30 C)(abs.) (g) (ppm) (ppm) Scale-up 1: 198 g 10% Crepetrol 870, 2 g 0.5 Mglycerol, 68 microliters HK1, 1.75 g nutrient solution. — 0 7.20 —  8.20ND 23 X33047-63-1 0.5 7.19 0.073 — ND 23 X33047-63-2 15.67 7.17 0.185 —ND 2.5 Scale-up 2: 150 g 18.73% Crepetrol 870, 50.0 g −63, 1.02 g ofAlcalase, 1.31 g nutrient solution. — 0 7.18 — 11.13 ND 32 X33047-65-1 17.17 0.181 — ND 32 X33047-65-2 4 7.17-7.19 0.207  0.13 ND 10 X33047-65-38 7.18 0.261 — 0.45 0.42 Scale-up 3: 150 g 18.73% Crepetrol 870, 50.0 g−65, 0.87 g of Alcalase, 1.31 g nutrient solution. — 0 7.21 — 11.26 ND32 X33047-67-1 0.083 7.21 0.193 — ND 31 X33047-67-2 13.75 7.17 0.288 —0.39 0.22 Scale-up 4: 266.7 g 18.73% Crepetrol 870, 133.3 g −67, 1.54 gof Alcalase, 2.33 g nutrient solution. — 0 7.22 — 20.28 ND 29X33047-69-1 1 7.20 0.194 — ND 24 X33047-69-2 4 7.20 0.234 — 0.45 2.7X33047-69-3 8 7.19 0.258 — 0.41 0.35 Scale-up 5: 200 g 18.73% Crepetrol870, 200.0 g −69, 1.16 g of Alcalase, 1.75 g nutrient solution. — 0 7.22— 15.08 ND 22 X33047-71-1 0.083 7.22 0.199 — ND 20 X33047-71-2 14.5 7.200.278 — 0.38 0.13 X33047-71 After Acid Test 0.30 1.5 Scale-up 6: 200 g18.73% Crepetrol 870, 200.0 g −71, 1.16 g of Alcalase, 1.75 g nutrientsolution. — 0 7.24 — 15.37 ND 22 X33047-73-1 1 7.22 0.224 — ND 16X33047-73-2 4 7.21 0.261 — 0.42 0.43 X33047-73-3 8 7.21 0.271 — 0.410.21 X33047-73 After Acid Test 0.32 3.3

Example 36 Biodehalogenation of Crepetrol® A6115 (see Table BP4 for Dataand Details)

[0454] A portion of Crepetol® A6115 creping agent without biocide(Available from Hercules Incorporated, Wilmington, Del.; Milwaukee, Wis.plant) was filtered through a 100 mesh screen. The resin had 15.73%total solids, a pH of 5.1 and a Brookfield viscosity of 86 cps.

[0455] Pasteurization: A 3-L round-bottom flask was fitted with acondenser, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 2770 g of the resin. The resin washeated over 1.5 hours from 25° C. to 85° C. The resin was held at 85° C.for 20 minutes and then cooled to 25° C. in 30 minutes. The pasteurizedresin was stored in a sterile container.

[0456] Biodehalogenation: Preparation of resin inoculum [Scale-up 1(SU1)]: A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. A portion of the pasteurized resin was diluted to10% with sterile, deionized water. To the flask was added 198 g of this10% resin and 2.0 g of 5 mM sterile glycerol in water solution. The pHwas raised to 6.0 with 1.04 g of 30% aqueous sodium hydroxide and then133 microliters of HK1 concentrated starter culture was added (1:1500,HK1 to resin) [See Example 24 for concentrated starter culturepreparation] and 1.75 g of a nutrient solution was added. (The nutrientsolution consisted of 8026 ppm of potassium dihydrogen phosphate, 27480ppm of urea, 4160 ppm of magnesium sulfate and 840 ppm of calciumchloride in tap water.) The air sparge was started, the temperature wasmaintained at 30° C. The bacterial growth was monitored by opticaldensity (OD₆₀₀) and the biodehalogenation was monitored by GC. OD₆₀₀ wasdetermined by measuring the optical density at a wavelength of 600 nmusing a Spectronic® Genesys™ UV/Vis spectrophotometer (SpectronicInstruments, Incorporated, Rochester, N.Y., USA) and a disposable cuvetwith 1-cm pathlength. After 16 hours, the resulting resin was used asinoculum for SU2.

[0457] Scale-up 2 (SU2)

[0458] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurizedresin. The pH was raised to 5.8 with 0.96 g of 30% aqueous sodiumhydroxide and then 50.0 g of the SU1 resin inoculum was added (25%inoculation rate) and 1.31 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 8 hours,the resulting resin was used as inoculum for SU3.

[0459] Scale-up 3 (SU3)

[0460] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurizedresin. The pH was raised to 5.8 with 0.96 g of 30% aqueous sodiumhydroxide and then 50.0 g of the SU2 resin inoculum was added (25%inoculation rate) and 1.31 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 14.5hours, the resulting resin was used as inoculum for SU4. The remainingresin not used for inoculum was discarded, but could have been used togive the finished product.

[0461] Scale-up 4 (SU4)

[0462] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurizedresin. The pH was raised to 5.8 with 1.40 g of 30% aqueous sodiumhydroxide and then 100.0 g of the SU3 resin inoculum was added (25%inoculation rate) and 2.62 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 8 hours,the resulting resin was used as inoculum for SU5. The remaining resinnot used for inoculum was discarded, but could have been used to givethe finished product.

[0463] Scale-up 5 (SU5)

[0464] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurizedresin. The pH was raised to 5.8 with 1.69 g of 30% aqueous sodiumhydroxide and then 100.0 g of the SU4 resin inoculum was added (25%inoculation rate) and 2.62 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 15 hours,the resulting resin was used as inoculum for SU6. The remaining resinnot used for inoculum was converted to finished product by lowering thepH to 5.3 with concentrate (96%) sulfuric acid and adding 2000 ppm ofpotassium sorbate as biocide.

[0465] Scale-up 6 (SU6)

[0466] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurizedresin. The pH was raised to 5.8 with 1.82 g of 30% aqueous sodiumhydroxide and then 100.0 g of the SU5 resin inoculum was added (25%inoculation rate) and 2.62 g of a nutrient solution was added. (Thenutrient solution consisted of 8026 ppm of potassium dihydrogenphosphate, 27480 ppm of urea, 4160 ppm of magnesium sulfate and 840 ppmof calcium chloride in tap water.) The air sparge was started, thetemperature was maintained at 30° C. The bacterial growth was monitoredby optical density (OD₆₀₀) and the biodehalogenation was monitored byGC. OD₆₀₀ was determined by measuring the optical density at awavelength of 600 nm using a Spectronic® Genesys™ UV/Visspectrophotometer (Spectronic Instruments, Incorporated, Rochester,N.Y., USA) and a disposable cuvet with 1-cm pathlength. After 8 hours,the resulting resin was converted to finished product by lowering the pHto 5.3 with 0.73 g of concentrated (96%) sulfuric acid and adding 2000ppm of potassium sorbate (7.6 mL of 10 wt % aqueous potassium sorbate)as biocide.

[0467] See Table 27 for the results from monitoring the treatment. TABLE27 Time Gardner OD₆₀₀ 30% DCP CPD Sample (hours) pH (30 C) Viscosity(abs.) NaOH (g) (ppm) (ppm) Scale-up 1: 198 g 10% Crepetrol A6115(pasteurized), 2 g 0.5 M glycerol, 133 microliters HK1 (1:1500,resin:inoculum), 1.75 g nutrient solution. — 0 5.96 — — 1.04 1.7 30X32989-58-1 0.25 5.95 — 0.059 — ND 30 X32989-58-2 16 5.95 — 0.355 — 1.20.28 Scale-up 2: 150 g 15.7% Crepetrol A6115 (pasteurized), 50.0 g −58,1.31 g nutrient solution. — 0 5.81 — — 0.96 2.0 35 X32989-60-1 1 5.81 —0.134 — 2.0 0.50 X32989-60-2 4 5.82 — 0.130 — 1.9 0.42 X32989-60-3 85.83 — 0.124 — 1.9 0.58 Scale-up 3: 150 g 15.7% Crepetrol A6115(pasteurized), 50.0 g −60, 1.31 g nutrient solution. — 0 5.81 — — 0.962.0 35 X32989-62-1 0.083 5.81 — 0.080 — ND 28 X32989-62-2 14.5 5.81 —0.078 — 2.8 0.59 Scale-up 4: 300 g 15.7% Crepetrol A6115 (pasteurized),100.0 g −62, 2.62 g nutrient solution. — 0 5.78 — — 1.40 2.0 35X32989-64-1 1 5.77 — 0.064 — ND 24 X32989-64-2 4 5.76 — 0.050 — 2.3 0.39X32989-64-3 8 5.77 — 0.055 — 2.3 0.49 Scale-up 5: 300 g 15.7% CrepetrolA6115 (pasteurized), 100.0 g −64, 2.62 g nutrient solution. — 0 5.80 — —1.69 2.0 35 X32989-66-1 0.083 5.76 E 0.054 — ND 33 X32989-66-2 15 5.76 E0.042 — 2.3 0.55 Scale-up 6: 300 g 15.7% Crepetrol A6115 (pasteurized),100.0 g −66, 2.62 g nutrient solution. — 0 5.77 — — 1.82 2.0 35X32989-68-1 1 5.76 E 0.036 — ND 24 X32989-68-2 4 5.75 — 0.054 — 2.1 1.7X32989-68-3 8 5.75 E 0.039 — 2.2 0.41

Example 37 Alcalase-Biodehalogenation of Crepetrol® A6115 creping agent(see Table BP5 for Data and Details)

[0468] A portion of Crepetol® A6115 creping agent without biocide(Available from Hercules Incorporated, Wilmington, Del.; Milwaukee, Wis.plant) was filtered through a 100 mesh screen. The resin had 15.73%total solids, a pH of 5.1 and a Brookfield viscosity of 86 cps.

[0469] Pasteurization: A 3-L round-bottom flask was fitted with acondenser, a temperature controlled circulating bath and a mechanicalstirrer. To the flask was added 2770 g of the resin. The resin washeated over 1.5 hours from 25° C. to 85° C. The resin was held at 85° C.for 20 minutes and then cooled to 25° C. in 30 minutes. The pasteurizedresin was stored in a sterile container.

[0470] Biodehalogenation: Preparation of resin inoculum [Scale-up 1(SU1)]: A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. A portion of the pasteurized resin was diluted to10% with sterile, deionized water. To the flask was added 198 g of this10% resin and 2.0 g of 5 mM sterile glycerol in water solution. The pHwas raised to 7.2 with 2.65 g of 30% aqueous sodium hydroxide and then0.62 g of Alcalase 2.5L type DX (available from Novozymes) and 133microliters of HK1 concentrated starter culture was added (1:1500, HK1to resin) [See Example 24 for concentrated starter culture preparation]and 1.75 g of a nutrient solution was added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 16 hours, the resulting resin was used as inoculum forSU2.

[0471] Scale-up 2 (SU2)

[0472] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurizedresin. The pH was raised to 7.2 with 3.37 g of 30% aqueous sodiumhydroxide and then 0.73 g of Alcalase 2.5L type DX (available fromNovozymes), 50.0 g of the SU1 resin inoculum (25% inoculation rate) and1.31 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 8 hours, the resulting resin was used as inoculum forSU3.

[0473] Scale-up 3 (SU3)

[0474] A 250-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 150 g of the pasteurizedresin. The pH was raised to 7.2 with 3.02 g of 30% aqueous sodiumhydroxide and then 0.73 g of Alcalase 2.5L type DX (available fromNovozymes), 50.0 g of the SU2 resin inoculum (25% inoculation rate) and1.31 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 14.5 hours, the resulting resin was used as inoculumfor SU4. The remaining resin not used for inoculum was discarded, butcould have been used to give the finished product.

[0475] Scale-up 4 (SU4)

[0476] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurized %resin. The pH was raised to 7.2 with 6.03 g of 30% aqueous sodiumhydroxide and then 1.46 g of Alcalase 2.5L type DX (available fromNovozymes), 100.0 g of the SU3 resin inoculum (25% inoculation rate) and2.62 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 8 hours, the resulting resin was used as inoculum forSU5. The remaining resin not used for inoculum was discarded, but couldhave been used to give the finished product.

[0477] Scale-up 5 (SU5)

[0478] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurizedresin. The pH was raised to 7.2 with 6.26 g of 30% aqueous sodiumhydroxide and then 1.46 g of Alcalase 2.5L type DX (available fromNovozymes), 100.0 g of the SU4 resin inoculum (25% inoculation rate) and2.62 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 15 hours, the resulting resin was used as inoculum forSU6. The remaining resin not used for inoculum was converted to finishedproduct by lowering the pH to 5.3 with concentrated (96%) sulfuric acidand adding 2000 ppm of potassium sorbate as biocide.

[0479] Scale-up 6 (SU6)

[0480] A 500-mL round-bottom flask was fitted with a condenser, a pHmeter, a temperature controlled circulating bath, an air sparge tube anda mechanical stirrer. To the flask was added 300 g of the pasteurizedresin. The pH was raised to 7.2 with 6.02 g of 30% aqueous sodiumhydroxide and then 1.46 g of Alcalase 2.5L type DX (available fromNovozymes), 100.0 g of the SU5 resin inoculum (25% inoculation rate) and2.62 g of a nutrient solution were added. (The nutrient solutionconsisted of 8026 ppm of potassium dihydrogen phosphate, 27480 ppm ofurea, 4160 ppm of magnesium sulfate and 840 ppm of calcium chloride intap water.) The air sparge was started, the temperature was maintainedat 30° C. The bacterial growth was monitored by optical density (OD₆₀₀)and the biodehalogenation was monitored by GC. OD₆₀₀ was determined bymeasuring the optical density at a wavelength of 600 nm using aSpectronic® Genesys™ UV/Vis spectrophotometer (Spectronic Instruments,Incorporated, Rochester, N.Y., USA) and a disposable cuvet with 1-cmpathlength. After 8 hours, the resulting resin was converted to finishedproduct by lowering the pH to 5.3 with 2.92 g of concentrated (96%)sulfuric acid and adding 2000 ppm of potassium sorbate (7.6 mL of 10 wt% aqueous potassium sorbate) as biocide. See Table 28 for the resultsfrom monitoring the treatment. TABLE 28 Scale-up 1: 198 g 10% CrepetrolA6115 (pasteurized), 2 g 0.5 M glycerol, 0.62 g of Alcalase, 133microliters HK1 (1:1500, resin:inoculum), 1.75 g nutrient solution. TimeGardner OD₆₀₀ 30% DCP CPD Sample (hours) PH (30 C) Viscosity (abs.) NaOH(g) (ppm) (ppm) — 0 7.17 — — 2.65 1.7 30 X32989-59-1 0.25 7.16 — 0.058 —ND 32 X32989-59-2 16 7.17 — 0.450 —  0.57 0.12 Time Gardner OD₆₀₀ 30%DCP CPD Sample (hours) pH (30 C) Viscosity (abs.) NaOH (g) (ppm) (ppm)Scale-up 2: 150 g 15.7% Crepetrol A6115, 0.73 g Alcalase, 50.0 g −59,1.31 g nutrient solution. — 0 7.19 — — 3.37 2.0 35 X32989-61-1 1 7.18 —0.167 — 1.7 3.6 X32989-61-2 4 7.19 — 0.180 — 1.5 0.27 X32989-61-3 8 7.18— 0.183 — 1.4 0.24 Scale-up 3: 150 g 15.7% Crepetrol A6115, 0.73 gAlcalase, 50.0 g −61, 1.31 g nutrient solution. — 0 7.17 — — 3.02 2.0 35X32989-63-1 0.083 7.16 — 0.087 — ND 33 X32989-63-2 14.5 7.16 — 0.130 —1.5 0.22 Scale-up 4: 300 g 15.7% Crepetrol A6115, 1.46 g Alcalase, 100.0g −63, 2.62 g nutrient solution. — 0 7.15 — — 6.03 2.0 35 X32989-65-1 17.13 — 0.079 — ND 31 X32989-65-2 4 7.12 — 0.095 — 1.9 0.59 X32989-65-3 87.11 — 0.110 — 1.9 0.37 Scale-up 5: 300 g 15.7% Crepetrol A6115, 1.46 gAlcalase, 100.0 g −65, 2.62 g nutrient solution. — 0 7.20 — — 6.26 2.035 X32989-67-1 0.083 7.17 C 0.050 — ND 35 X32989-67-2 15 7.17 C 0.107 —1.7 0.19 Scale-up 6: 300 g 15.7% Crepetrol A6115, 1.46 g Alcalase, 100.0g −67, 2.62 g nutrient solution. — 0 7.16 — — 6.02 2.0 35 X32989-69-1 17.14 C 0.061 — 2.5 32 X32989-69-2 4 7.13 — 0.099 — 1.9 1.1 X32989-69-3 87.13 C 0.107 — 1.8 0.23

Example 38 High Solids, Simultaneous Enzyme-Biodehalogenation Treatment

[0481] General Procedure

[0482] Low molecular weight terpolymers of adipic acid,diethylenetriamine and acetic acid were prepared by condensing thesereactants at 170° C. for three hours in a molar ratio of 1:0.9:0.2. Thereaction products were diluted to 50% solids.

[0483] These polymers were reacted with epichlorohydrin at anepichlorohydrin:diethylenetriame ratio of 0.82 for 3.5 hours at 40° C.,and water was added in such a way that the total solids content of thereactor was 40%.

[0484] In a next step, the reaction mixtures were diluted to a totalsolids content of 31% and heated to 68° C. for functionalization andcrosslinking. Reactions were stopped at Gardner-Holt viscosity “I/J”after about two hours and 10 minutes at this temperature by the additionof 30% sulfuric acid in such a way that the pH after sulfuric additionwas 4.5. The reaction products were cooled to room temperature, 1.75% ofphosphoric acid (weight to reactor volume) was added and the pH adjustedafter phosphoric acid addition using sulfuric acid to pH 2.7. Thepurpose of adding phosphoric and sulfuric acid to this pH is to obtainviscometrically stable resins.

[0485] The resins were analyzed for their residual organochlorine leveland 1,3-DCP levels were found to be 816 ppm for the acetic acidcontaining material. These resins were tested in a paper trial and werefound to be as effective as Kymene® SLX2 in imparting wet strength topaper. The resins were stored for 6 weeks at 32° C. and during thisperiod gelation of the resins did not occur.

[0486] Biodehalogenation (see Table 29 for data and details): Aninoculum was prepared with a non-end-capped resin (Kymene E7219) (seeScale-up 1 and Scale-up 2 in Table 29). The end-capped resin preparedabove was diluted to 13.5% solids, the pH was raised to pH 7.2 with 30%aqueous sodium hydroxide, the catalyst (Alcalase, Novozymes) forhydrolyzing the CPD-forming species, the inoculum from Scale-up 2, andthe nutrient solution were added. Not wishing to be bound by theory, itis believed that this low solids intermediate step is useful to improvethe adaptation of the microbial population to the new resin. Afterbiodehalogenation was complete, the next batch (Scale-up 4) was started.The end-capped resin prepared above was diluted to 20% solids, the pHwas raised to pH 7.2 with 30% aqueous sodium hydroxide, the catalyst(Alcalase, Novozymes) for hydrolyzing the CPD-forming species, theinoculum from Scale-up 3 (20% inoculation rate), and the nutrientsolution were added. The microbial growth was rapid, as indicated byoptical density (OD₆₀₀) (see Scale-up 4 in Table 29). Thebiodehalogenation was also rapid, as indicated by the rapid loss of DCPand CPD. TABLE 29 Alcalase-Biodehalogenation of a High SolidsWet-Strength Resin. Time pH @ Gardner OD₆₀₀ 30% DCP CPD Sample Time(hours) 30 C Viscosity (abs.) NaOH (g) (ppm) (ppm) Scale-up 1: 200 g 8%E7219 (pasteurized), No Alcalase, 400 microliters of HK7, 1.75 gnutrient solution. —  6:56 0 7.15 — — 2.54 — — X32966-3-1  7:58 1 7.14 —0.171 — 213 143 X32966-3-2 10:57 4 7.13 — 0.162 — 144 181 X32966-3-313:58 7 7.08- — 0.162 0.11  62 235 7.25 X32966-3-4 17:00 10 7.21 — 0.184— ND 274 X32966-3-5 20:10 13 7.17 — 0.204 — ND 228 X32966-3-6  5:55 237.03 — 0.509 — ND 2.6 Scale-up 2: 350 g 13.5% E7219 (pasteurized), 5.03g Alcalase, 87.5 g of −3, 3.06 g nutrient solution. —  6:40 0 7.23 — —7.31 — — X32966-5-1  7:40 1 7.20 — 0.113 —  80 391 X32966-5-2 10:41 47.04- — 0.149 0.33 ND 415 7.21 X32966-5-3 13:30 7 7.19 — 0.248 — ND 313X32966-5-4 16:50 10 7.11- — 0.383 0.31 ND 98 7.27 X32966-5-5 20:40 147.28 — 0.475 — ND 38 X32966-5-6  5:59 23 7.17 — 0.573 — ND 0.43 Thefinal resin had a Brookfield Viscosity of 38 cps. Scale-up 3: 200 g13.5% endcapped resin, 2.50 g Alcalase, 22.22 g of −5, 1.75 g nutrientsolution. —  7:00 0 7.22 — — 5.71 — — X32966-11-1  8:01 1 7.18 — 0.059 —270 343 X32966-11-2 11:02 4 7.05- — 0.092 0.28  40 621 7.23 X32966-11-314:01 7 7.17 — 0.165 — ND 660 X32966-11-4 17:00 10 7.15- — 0.277 0.15 ND627 7.31 X32966-11-5  5:50 23 7.11 — 0.772 — ND 0.4 Scale-up 4: 160 g20% endcapped resin, 3.00 g Alcalase, 40.0 g of −11, 1.40 g nutrientsolution. —  6:56 0 7.25 — — 6.09 — — X32966-15-1  8:00 1 7.21 A-B 0.148— 406 393 X32966-15-2 12:15 5.3 7.06- A-A-1 0.346 0.27 283 586 7.23X32966-15-3 17:45 10.6 7.03- A-A-1 0.728 0.23  86 14 7.16 X32966-15-4 9:30 26.5 7.06 A-A-1 0.962 — ND 0.68 X32966-15-5  8:30 49.5 6.91 A-A-11.004 — ND 0.22

We claim:
 1. A process for rendering a polyamine-epihalohydrin resinstorage stable, comprising: treating a composition containing a wetstrength polyamine-epihalohydrin resin, the composition comprising asolids content of at least 15 wt % and including CPD-forming species,with at least one enzymatic agent under conditions to at least one ofinhibit, reduce and remove the CPD-forming species to obtain a gelationstorage stable reduced CPD-forming resin so that the compositioncontaining the reduced CPD-forming polyamine-epihalohydrin resin whenstored for 24 hours at 50° C., and a pH of about 1.0 releases less thanabout 250 ppm dry basis of CPD.
 2. The process according to claim 1,wherein the composition containing the reduced CPD-formingpolyamine-epihalohydrin resin when stored for 24 hours at 50° C., and apH of about 1.0 releases less than about 50 ppm dry basis of CPD.
 3. Theprocess according to claim 1, wherein the treatment conditions comprisea temperature of from about 20° C. to 60° C.
 4. The process according toclaim 3, wherein the treatment conditions comprise a temperature of fromabout 20° C. to 40° C.
 5. The process according to claim 1, wherein thetreatment conditions comprise a reaction time of from about 30 minutesto about 96 hours.
 6. The process according to claim 5, wherein thetreatment conditions comprise a reaction time of from about 2 hours toabout 12 hours.
 7. The process according to claim 1, wherein thetreatment conditions comprise a pH of from about 2.5 to about
 9. 8. Theprocess according to claim 7, wherein the treatment conditions comprisea pH of from about 7 to about
 9. 9. The process according to claim 8,wherein the treatment conditions comprise a pH of from about 6 to about8.5.
 10. The process according to claim 1, wherein the ratio of at leastone enzymatic agent to polyamine-epihalohydrin resin (dry basis) is fromabout 1:1600 to about 1:1.5.
 11. The process according to claim 10,wherein the ratio of at least one enzymatic agent topolyamine-epihalohydrin resin (dry basis) is from about 1:160 to about1:4.
 12. The process according to claim 1, wherein the ratio of at leastone enzymatic agent (active enzyme, dry basis) topolyamine-epihalohydrin resin (dry basis) is from about 0.04:1600 toabout 0.04:1.5.
 13. The process according to claim 1, wherein the solidscontent is 15 to 50 wt % active solids, the treatment conditionscomprise a temperature of from about 0° C. to about 35° C., a reactiontime of from about 4 to about 24 hours, a pH of from about 6.9 to about7.9, the ratio of at least one enzymatic agent topolyamine-epihalohydrin resin (dry basis) is from about 1:20 to about1:8.
 14. The process according to claim 1, wherein the at least oneenzymatic agent is selected from the group consisting of an esterase, alipase, a protease or a combination thereof.
 15. The process accordingto claim 1, wherein the at least one enzymatic agent is a protease inthe subtilisin group.
 16. The process according to claim 1, wherein theat least one enzymatic agent has esterase activity.
 17. The processaccording to claim 1, wherein the at least one enzymatic agent isproduced from a microorganism selected from the group consisting ofBacillus licheniformis (Swiss-Prot Accession Number: P00780), orBacillus amyloliquifaciens (P00782), and Bacillus lentus (P29600). 18.The process according to claim 1, wherein the at least one enzymaticagent is ALCALASE.
 19. The process according to claim 1, wherein theresin is characterized by the presence of the functionality representedby the formula:


20. The process according to claim 1, wherein the resin is characterizedby the presence of the functionality represented by the formula:


21. The process according to claim 1, wherein the resin is characterizedby the presence of the functionality represented by the formula:

wherein X⁻ is an anion.
 22. The process according to claim 1, wherein,at least one of simultaneously with, prior to or subsequent to thetreating a composition containing polyamine-epihalohydrin resin toobtain a reduced CPD-forming resin, the resin is contacted with at leastone microorganism, or at least one enzyme isolated from the at least onemicroorganism, in an amount, and at a pH and temperature effective todehalogenate residual quantities of organically bound halogen.
 23. Theprocess according to claim 22 wherein the at least one microorganism, orat least one enzyme isolated from the at least one microorganism is ahydrogen halide lysase type dehalogenase.
 24. The process according toclaim 22 wherein the at least one microorganism, or at least one enzymeisolated from the at least one microorganism comprises at least one ofArthrobacter histidinolovorans (HK1), and Agrobacterium radiobacter(HK7).
 25. The process according to claim 22, wherein the at least onemicroorganism comprises a mixture comprising at least one ofAgrobacterium radiobacter (HK7) and, Arthrobacter histidinolovorans(HK1).
 26. The process according to claim 1, wherein, simultaneouslywith the treating a composition containing polyamine-epihalohydrin resinto obtain a reduced CPD-forming resin, the CPD-forming resin iscontacted with at least one microorganism, or at least one enzymeisolated from the at least one microorganism, in an amount, and at a pHand temperature effective to dehalogenate residual quantities oforganically bound halogen.
 27. The process according to claim 26,wherein the treatment conditions comprise a reaction time of 48 hours orless.
 28. The process according to claim 26, wherein the temperature offrom about 20° C. to 35° C.
 29. The process according to claim 26,wherein the treatment conditions comprise a pH of from about 6.5 to 8.0.32. The process according to claim 26 wherein the ratio of at least oneenzymatic agent to polyamine-epihalohydrin resin (dry basis) is fromabout 1:1600 to about 1:1.5.
 30. The process according to claim 26wherein the at least one microorganism, or at least one enzyme isolatedfrom the at least one microorganism is a hydrogen halide lysase typedehalogenase.
 31. The process according to claim 26 wherein the at leastone microorganism, or at least one enzyme isolated from the at least onemicroorganism comprises at least one of Arthrobacter histidinolovorans(HK1), and Agrobacterium radiobacter (HK7).
 32. The process according toclaim 26, wherein the at least one microorganism comprises a mixturecomprising at least one of Agrobacterium radiobacter (HK7) and,Arthrobacter histidinolovorans (HK1).
 33. The process according to claim26 wherein the treatment conditions comprise a reaction time of 48 hoursor less, a temperature of from about 20° C. to 35° C., a pH of fromabout 6.5 to about 8.0 and the ratio of at least one enzymatic agent topolyamine-epihalohydrin resin (dry basis) is from about 1:1600 to about1:1.5 and the at least one microorganism comprises a mixture comprisingat least one of Agrobacterium radiobacter (HK7) and, Arthrobacterhistidinolovorans (HK1).
 34. The process according to claim 1, wherein,simultaneously, prior to or subsequent to the treating a compositioncontaining polyamine-epihalohydrin resin to obtain a reduced CPD-formingresin, the resin is treated to reduce at least one of epihalohydrins,epihalohydrin hydrolysis by-products and organic halogen bound to thepolymer backbone.
 35. A process for preparing a paper product,comprising: treating a composition containing wet strengthpolyamine-epihalohydrin resin, the composition comprising a solidscontent of at least 15 wt % and including CPD-forming species, with atleast one enzymatic agent under conditions to at least one of inhibit,reduce and remove the CPD-forming species to obtain a gelation storagestable reduced CPD-forming resin, and forming a paper product with thereduced CPD-forming polyamine-epihalohydrin resin, so that a paperproduct, when corrected for adding at about a 1 wt % addition level ofthe reduced CPD-forming resin, contains less than about 250 ppb of CPD.36. The process according to claim 35, wherein the paper product, whencorrected for adding at about a 1 wt % addition level of the reducedCPD-forming resin, contains less than about 50 ppb of CPD.
 37. Theprocess according to claim 35, wherein the solids content is 15 to 50 wt% active solids, the temperature of the reaction is from about 0° C. toabout 35° C., the reaction time is from about 4 to about 24 hours andthe pH of the reaction is from about 6.9 to about 7.9, the ratio of atleast one enzymatic agent to polyamine-epihalohydrin resin (dry basis)is from about 1:20 to about 1:8.